Gene upregulated in cancers of the prostate

ABSTRACT

The present invention relates to a novel protein designated 20P2H8 which shares homology with several heterogenous nuclear ribonucleoproteins (hnRNPs). A full length approximately 3600 bp 20P2H8 cDNA (SEQ ID NO: 10, encoding a 517 amino acid open reading frame (SEQ ID NO: 2), is provided herein.

This application claims the benefit of U.S. provisional patentapplication No. 60/162,364, filed Oct. 28, 1999, now abandoned, theentire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed 20P2H8, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers which express20P2H8, particularly including cancers of the bladder, prostate, colonand pancreas.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people each year, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine, still very much in its infancy, promisesto redefine the ways in which these cancers are managed. Unquestionably,there is an intensive worldwide effort aimed at the development of novelmolecular approaches to cancer diagnosis and treatment. For example,there is a great interest in identifying truly tumor-specific genes andproteins that could be used as diagnostic and prognostic markets and/ortherapeutic targets or agents. Research efforts in these areas areencouraging, and the increasing availability of useful moleculartechnologies has accelerated the acquisition of meaningful knowledgeabout cancer. Nevertheless, progress is slow and generally uneven.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis more or less the same for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy remain fixed as the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with significant undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects, as furtherdiscussed below. Most prostate cancers initially occur in the peripheralzone of the prostate gland, away from the urethra. Tumors within thiszone may not produce any symptoms and, as a result, most men withearly-stage prostate cancer will not present clinical symptoms of thedisease until significant progression has occurred. Tumor progressioninto the transition zone of the prostate may lead to urethralobstruction, thus producing the first symptoms of the disease. However,these clinical symptoms are indistinguishable from the commonnon-malignant condition of benign prostatic hyperplasia (BPH). Earlydetection and diagnosis of prostate cancer currently relies on digitalrectal examinations (DRE), prostate specific antigen (PSA) measurements,transrectal ultrasonography (TRUS), and transrectal needle biopsy(TRNB). At present, serum PSA measurement in combination with DRErepresent the leading tool used to detect and diagnose prostate cancer.Both have major limitations which have fueled intensive research intofinding better diagnostic markers of this disease.

Similarly, there is no available marker that can predict the emergenceof the typically fatal metastatic stage of prostate cancer. Diagnosis ofmetastatic stage is presently achieved by open surgical or laparoscopicpelvic lymphadenectomy, whole body radionuclide scans, skeletalradiography, and/or bone lesion biopsy analysis. Clearly, better imagingand other less invasive diagnostic methods offer the promise of easingthe difficulty those procedures place on a patient, as well as improvingdiagnostic accuracy and opening therapeutic options. A similar problemis the lack of an effective prognostic marker for determining whichcancers are indolent and which ones are or will be aggressive. PSA, forexample, fails to discriminate accurately between indolent andaggressive cancers. Until there are prostate tumor markers capable ofreliably identifying early-stage disease, predicting susceptibility tometastasis, and precisely imaging tumors, the management of prostatecancer will continue to be extremely difficult.

PSA is the most widely used tumor marker for screening, diagnosis, andmonitoring prostate cancer today. In particular, several immunoassaysfor the detection of serum PSA are in widespread clinical use. Recently,a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSAmRNA in serum has been developed. However, PSA is not a disease-specificmarker, as elevated levels of PSA are detectable in a large percentageof patients with BPH and prostatitis (25-86%)(Gao et al., 1997, Prostate31: 264-281), as well as in other nonmalignant disorders and in somenormal men, a factor which significantly limits the diagnosticspecificity of this marker. For example, elevations in serum PSA ofbetween 4 to 10 ng/ml are observed in BPH, and even higher values areobserved in prostatitis, particularly acute prostatitis. BPH is anextremely common condition in men. Further confusing the situation isthe fact that serum PSA elevations may be observed without anyindication of disease from DRE, and visa-versa. Moreover, it is nowrecognized that PSA is not prostate-specific (Gao et al., supra, forreview).

Various methods designed to improve the specificity of PSA-baseddetection have been described, such as measuring PSA density and theratio of free vs. complexed PSA. However, none of these methodologieshave been able to reproducibly distinguish benign from malignantprostate disease. In addition, PSA diagnostics have sensitivities ofbetween 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), andthus miss identifying prostate cancer in a significant population of menwith the disease.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54: 1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252) andprostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95: 1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su et al, 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

SUMMARY OF THE INVENTION

The present invention relates to the gene designated 20P2H8, which isover-expressed in cancers including cancer of the prostate. Northernblot expression analysis of 20P2H8 gene expression in normal tissuesshows a predominant transcript of about 4.4 Kb that is highly expressedin pancreas, and is also expressed in prostate, colon and placenta. Thepredicted molecular weight of the 20P2H8 protein is 57.4 kD and its' pIis 7.7. In addition, expression analysis demonstrates high levels of20P2H8 expression in several prostate and other cancer cell lines aswell as prostate, bladder, kidney and breast cancer patient samples andtumor xenografts. The expression profile of 20P2H8 in normal adulttissues, combined with the over-expression observed in cancer cells suchas pancreas, bladder, testis, lung, ovary and prostate cancer cell linesand/or cancer patient samples, provides evidence that 20P2H8 isaberrantly expressed in at least some cancers, and can serve as a usefuldiagnostic and/or therapeutic target for such cancers.

The invention provides polynucleotides corresponding or complementary toall or part of the 20P2H8 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding 20P2H8proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to the20P2H8 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 20P2H8 genes, mRNAs, or to20P2H8-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 20P2H8. Recombinant DNA moleculescontaining 20P2H8 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 20P2H8gene products are also provided. The invention further provides 20P2H8proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to 20P2H8 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker.

The invention further provides methods for detecting the presence andstatus of 20P2H8 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 20P2H8. Atypical embodiment of this invention provides methods for monitoring20P2H8 gene products in a tissue sample having or suspected of havingsome form of growth dysregulation such as cancer.

The invention further provides various therapeutic compositions andstrategies for treating cancers that express 20P2H8 such as prostatecancers, including therapies aimed at inhibiting the transcription,translation, processing or function of 20P2H8 as well as cancervaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show the nucleotide (SEQ ID NO: 1) and deduced amino acid(SEQ ID NO: 2) sequences of 20P2H8 cDNA. The start methionine andputative Kozak sequence are indicated in bold, RNA-binding domains RNP1and RNP2 (shaded) are boxed, 3 Proline-rich regions are in bold.

FIGS. 2A-2C show a semi-quantitative RT-PCR analysis of 20P2H8 geneexpression in a panel of 16 normal human tissues (Panels 2B and 2C) andseveral prostate cancer xenografts (Panel 2A). Lanes 1-8 in panel 2Acorrespond to RT-PCR analysis of 20P2H8 gene expression in brain,prostate, LAPC-4 AD, LAPC-4 AI, LAPC-9 AD, HeLa, mouse mix and anegative control respectively. Lanes 1-8 in panel 2B correspond toRT-PCR analysis of 20P2H8 gene expression in brain, heart, kidney,liver, lung, pancreas, placenta and skeletal muscle respectively. Lanes1-8 in panel 2C correspond to RT-PCR analysis of 20P2H8 gene expressionin colon, ovary, leukocytes, prostate, small intestine, spleen, testisand thymus respectively. The RT-PCR primers are specific for 20P2H8 andare as follows: 5′-TCTTGAAACCTCCAGACACAAGAA-3′ and5′-AAGTTACGATTTGGCTTCACTGG-3′ (SEQ ID NOS: 13 and 16, respectively). The20P2H8 PCR product is expected to be of 162 base pairs as marked by anarrow.

FIGS. 3A-3C show a Northern blot analysis of human 20P2H8 expression invarious normal tissues showing predominant expression in pancreas withsignificant expression also detected in prostate and colon. Lanes 1-8 inpanel A correspond to Nothern analysis of 20P2H8 gene expression inheart, brain, placenta, lung, liver, skeletal muscle, kidney andpancreas respectively. Lanes 1-8 in panel B correspond to Nothernanalysis of 20P2H8 gene expression in spleen, thymus, prostate, testis,ovary, small intestine, colon and leukocytes respectively. Lanes 1-5 inpanel C correspond to Nothern analysis of 20P2H8 gene expression inprostate, LAPC-4 AD, LAPC-4 AI, LAPC-9AD and LAPC-9 AI respectively.

FIG. 4 shows a Northern blot analysis of human 20P2H8 mRNA expression ina panel of human colon and pancreatic cancer cell lines.

FIGS. 5A-5B show a Northern expression analysis of 20P2H8 mRNA in manycancer lines including bladder, lung, breast, testicular cervical andovarian cancers.

FIG. 6 illustrates an RT-PCR analysis showing expression of the of20P2H8 mRNA in bladder cancer, colon cancer, and lung cancer patients.

FIGS. 7A-7B show expression of recombinant 20P2H8 protein. A. 293T cellswere transiently transfected with either pCDNA3.1 MycHis tagged 20P2H8plasmid or with empty control vector and harvested 2 days later. Cellswere lysed in SDS-PAGE sample buffer and lysates were separated on a10-20% SDS-PAGE gel and then transferred to nitrocellulose. The blot wasblocked in Tris-buffered saline (TBS)+2% non-fat milk and then probedwith a 1:3,000 dilution of rabbit anti-His pAb (Santa Cruz BiotechnologyInc.) in TBS+0.15% Tween-20+1% milk. The blot was washed and thenincubated with a 1:4,000 dilution of anti-rabbit HRP conjugate secondaryantibody. Following washing, anti-His immunoreactive bands weredeveloped by enhanced chemiluminescence and visualized by exposure toautoradiographic film. Indicated by arrow is a specific anti-Hisimmunoreactive band of approximately 60 Kd that corresponds toexpression of the Myc/His-tagged 20P2H8 protein in the transfectedcells. B. Cell lysates of 20P2H8 mRNA negative 293T cells and of 20P2H8mRNA positive Colo 205 cells were separated by SDS-PAGE and subjected toWestern blot analysis as indicated above but using an anti-20P2H8affinity purified polyclonal antibody (4 ug/ml). Indicated with arrowsare anti-20P2H8 immunoreactive bands of approximately 58 kD and 30 kDthat appear in 20P2H8 mRNA positive Colo 205 cells but not in 20P2H8mRNA negative 293T cells.

FIG. 8 shows a Northern analysis from tissues from patients with bladdercancer and unaffected individuals. Northern analysis was performed onbladder cancer and their adjacent normal matched tissues obtained fromfour bladder cancer patients. For this experiment, 10 μg of total RNAwas loaded for each sample. Overexpression of 20P2H8 was seen in 3 outof 4 tumor samples tested. No expression was observed in any of the 3normal matched tissues tested, nor in bladder isolated from a normalindividual. The data demonstrate specific expression of 20P2H8 in cancerbut not in normal bladder tissues.

FIG. 9 shows a RNA dot blot analysis from tissues from patients withvarious cancers. Expression of 20P2H8 was assayed in a panel of humancancers (T) and their respective matched normal tissues (N) on RNA dotblots. 20P2H8 overexpression was detected in 4 out of 14 kidney cancers,7 out of 9 breast cancer 1 out of 3 prostate cancers, 2 out of 3 ovariancancers, 1 out of 1 cervical cancer, and 4 out of 8 stomach cancers.20P2H8 was also found to be highly expressed in the melanoma cell lineG361 and the colorectal adenocarcinoma line SW480.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning. A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers which have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage TxNxM+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least about 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least about6 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C., and most preferably tostringent hybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium. citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative positions that are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions that are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. For example, % identity values may begenerated by WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology266:460-480; http://blast.wust1/edu/blast/README.html). Further detailsregarding amino acid substitutions, which are considered conservativeunder such criteria, are provided below. Additional definitions areprovided throughout the subsections that follow.

As discussed in detail below, the present invention relates to a novelprotein designated 20P2H8 which shares homology with severalheterogenous nuclear ribonucleoproteins (hnRNPs). A full lengthapproximately 3600 bp 20P2H8 cDNA (SEQ ID NO: 1), encoding a 517 aminoacid open reading frame (SEQ ID NO: 2), is provided herein. Thepredicted 20P2H8 protein exhibits five RNA binding sequences, two ofwhich correspond to ribonucleoprotein-1 (RNP1) consensus sites, andthree of which correspond to RNP2 sites. In addition, 20P2H8 containsthree regions with significant proline content (30-42%), which lieoutside regions of homology with hnRNPs. These proline rich regions maybe involved in protein-protein interactions as has been observed inother proteins (Schlessenger et al., 1994, Curt. Opin. Genet. Dev. 4:25). The 20P2H8 protein structure shows highest homology with a proteinidentified in C. elegans (Genbank CAA92704), and significant homology isalso seen with various hnRNPs involved in mRNA splicing (ROF, ROH1 andROH2; Honore et al., 1995, J. Biol. Chem. 270: 28780). Accordingly,based on these structural features and homologies, it is likely that the20P2H8 protein is involved in RNA splicing.

Expression analysis by northern blot shows highest expression levels ofa single 4.4 kb 20P2H8 transcript in pancreas, and is also expressed inprostate, colon and placenta. No other normal tissues in this panel showdetectable expression. Expression of 20P2H8 is up-regulated in humanprostate tumor xenografts derived from metastatic prostate cancer(compared to normal prostate). Further, analysis of 20P2H8 expression inmultiple cancer cell lines reveals high levels of expression in severalpancreatic (BxPC-3, HPAC, Capan-1) and colon (CaCo-2, T84, Colo-205)cancer cell lines. The up-regulated expression of 20P2H8 in cancersincluding cancers of the bladder, kidney, prostate, pancreas and colonindicates that the 20P2H8 gene, message and protein allows 20P2H8 to beused as a diagnostic, staging and/or prognostic marker for cancers ofthe prostate, colon and pancreas, and/or may also serve as a target forvarious approaches to the treatment of these cancers.

The invention provides polynucleotides corresponding or complementary toall or part of the 20P2H8 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding 20P2H8proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to the20P2H8 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides which hybridize to the 20P2H8 genes, mRNAs, or to20P2H8-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 20P2H8. Recombinant DNA moleculescontaining 20P2H8 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 20P2H8gene products are also provided. The invention further provides 20P2H8proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to 20P2H8 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, antibodies labeled with a detectable marker,and antibodies conjugated to radionuclides, toxins or other therapeuticcompositions. The invention further provides methods for detecting thepresence of 20P2H8 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express a 20P2H8.The invention further provides various therapeutic compositions andstrategies for treating cancers which express 20P2H8 such as prostateand bladder cancers, including antibody, vaccine and small moleculetherapy, and therapies aimed at inhibiting the transcription,translation, processing or function of 20P2H8.

Molecular Biology of 20P2H8

As is further described in the Examples that follow, the 20P2H8 gene andprotein have been characterized using a number of analytical approaches.For example, analyses of nucleotide coding and amino acid sequences wereconducted in order to identify potentially related molecules, as well asrecognizable structural domains, topological features, and otherelements within the 20P2H8 mRNA and protein structure. Northern blotanalyses of 20P2H8 mRNA expression were conducted in order to establishthe range of normal and cancerous tissues expressing 20P2H8 message.

The isolated 20P2H8 cDNA (SEQ ID NO: 1) provided herein (FIG. 1) andcorresponding gene are predicted to encode a 517 amino acid protein (SEQID NO: 2) with various structural features common to heterogenousnuclear ribonucleoproteins (hnRNPs) involved in RNA splicing, includingfive RNA binding sequences (two corresponding to ribonucleoprotein-1(RNP1) and 3 corresponding to ribonucleoprotein-2 (RNP2). The proteinexhibits significant homology to a C. elegans protein, designatedCAA92704 (approximately 52.4% identity in a 311 residues overlapbeginning with 20P2H8 amino acid residue 63 as shown in FIG. 1), as wellas several hnRNPs involved in RNA splicing (e.g., ROF, ROH1 and ROH;Honore et al., 1995, J. Biol. Chem. 270: 28780). In addition, the 20P2H8protein contains three regions with significant proline content(30-42%), which lie outside its regions of homology with hnRNPs. Theseproline rich regions may be involved in protein-protein interactions ashas been observed in other proteins (Schlessenger et al., 1994, Curr.Opin. Genet. Dev. 4: 25).

The 20P2H8 gene is normally expressed predominantly in pancreas, withlower levels of expression occurring in prostate, colon and placenta(FIGS. 2 and 3), but is also expressed or over-expressed in a number ofhuman cancers, including cancers of the prostate, kidney, skin, stomach,cervix, bladder, testis, ovaries, breast, pancreas, colon and lung (seee.g. FIGS. 4-8).

20P2H8 overexpression in the prostate cancer xenografts as well askidney, stomach, breast and is an indication that this molecule isderegulated in prostate cancer. Therefore, 20P2H8 target for prostatecancer diagnosis and therapy. For example, interfering with 20P2H8function, using an antibody and/or a small molecule, in prostate cancercells may prevent de-differentiation and/or proliferation of cells. Inaddition, small molecules and/or specific antibodies may interfere with20P2H8 function making this molecule a candidate for vaccinemethodologies. Investigating 20P2H8 function may also lead toidentification of other potential targets.

20P2H8 function can be assessed in mammalian cells using a variety oftechniques that are well known in the art. For mammalian expression,20P2H8 can be cloned into several vectors, including pcDNA 3.1myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller etal., 1991, MCB 11:1785). Using these expression vectors, 20P2H8 can beexpressed in several cell lines, including PC-3, NIH 3T3, LNCaP and293T. Expression of 20P2H8 can be monitored using northern blotanalysis.

The mammalian cell lines expressing 20P2H8 can be tested in several invitro and in vivo assays, including cell proliferation in tissueculture, activation of apoptotic signals, tumor formation in SCID mice,and in vitro invasion using a membrane invasion culture system (MICS)(Welch et al., Int. J. Cancer 43: 449-457). The 20P2H8 cell phenotypecan be compared to the phenotype of cells that lack expression of20P2H8.

As disclosed herein, 20P2H8 exhibits specific properties that areanalogous to those found in a family of genes whose polynucleotides,polypeptides and anti-polypeptide antibodies are used in well knowndiagnostic assays directed to examining conditions associated withdysregulated cell growth such as cancer, in particular prostate cancer(see e.g. both its highly specific pattern of tissue expression as wellas its overexpression in prostate cancers as described for example inExample 3). The best known member of this class is PSA, the archetypalmarker that has been used by medical practitioners for years to identifyand monitor the presence of prostate cancer (see e.g. Merrill et al., J.Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol.Aug.;162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst.91(19): 1635-1640(1999)). A variety of other diagnostic markers are alsoused in this context including p53 and K-ras (see e.g. Tulchinsky etal., Int J Mol Med 1999 Jul.;4(1):99-102 and Minimoto et al., CancerDetect Prev 2000;24(1):1-12). Consequently, this disclosure of the20P2H8 polynucleotides and polypeptides (as well as the 20P2H8polynucleotide probes and anti-20P2H8 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 20P2H8polynucleotides, polypeptides and antibodies described herein areanalogous to those methods from well established diagnostic assays whichemploy PSA polynucleotides, polypeptides and antibodies. For example,just as PSA polynucleotides are used as probes (for example in Northernanalysis, see e.g. Sharief et al., Biochem. Mol. Biol. Int.33(3):567-74(1994)) and primers (for example in PCR analysis, see e.g.Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe thepresence and/or the level of PSA mRNAs in methods of monitoring PSAoverexpression or the metastasis of prostate cancers, the 20P2H8polynucleotides described herein can be utilized in the same way todetect 20P2H8 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods ofmonitoring PSA protein overexpression (see e.g. Stephan et al., Urology5(4):560-3 (2000)) or the metastasis of prostate cells (see e.g. Alanenet al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 20P2H8polypeptides described herein can be utilized to generate antibodies foruse in detecting 20P2H8 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the bladder, kidney or prostate glandetc.) to a different area of the body (such as a lymph node), assayswhich examine a biological sample for the presence of cells expressing20P2H8 polynucleotides and/or polypeptides can be used to provideevidence of metastasis, for example, when a biological sample fromtissue that does not normally contain 20P2H8 expressing cells (lymphnode) is found to contain 20P2H8 expressing cells such as the 20P2H8expression seen in LAPC4 and LAPC9, xenografts isolated from lymph nodeand bone metastasis respectively. Alternatively 20P2H8 polynucleotidesand/or polypeptides can be used to provide evidence of cancer, forexample, when a cells in biological sample that do not normally express20P2H8 or express 20P2H8 at a different level (such as kidney, bladder,lung and prostate cells etc.) are found to express 20P2H8 or have anincreased expression of 20P2H8. In such assays, artisans may furtherwish to generate supplementary evidence of metastasis by testing thebiological sample for the presence of a second tissue restricted marker(in addition to 20P2H8) such as PSA, PSCA etc. (see e.g. Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring thismolecule, 20P2H8 polynucleotide fragments and polynucleotide variantscan also be used in an analogous manner. In particular, typical PSApolynucleotides used in methods of monitoring this molecule are probesor primers which consist of fragments of the PSA cDNA sequence.Illustrating this, primers used to PCR amplify a PSA polynucleotide mustinclude less than the whole PSA sequence to function in the polymerasechain reaction. In the context of such PCR reactions, skilled artisansgenerally create a variety of different polynucleotide fragments thatcan be used as primers in order to amplify different portions of apolynucleotide of interest or to optimize amplification reactions (seee.g. Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998);Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additionalillustration of the utility of such fragments is provided in Example 3,where a 20P2H8 polynucleotide fragment is used as a probe to show theoverexpression of 20P2H8 mRNAs in cancer cells. In addition, in order tofacilitate their use by medical practitioners, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see e.g. Sawai et al., Fetal Diagn.Ther. 1996 Nov.-Dec.;11(6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds., 1995)).Polynucleotide fragments and variants are typically useful in thiscontext as long as they have the common attribute or characteristic ofbeing capable of binding to a target polynucleotide sequence (e.g. the20P2H8 polynucleotide shown in SEQ ID NO: 1) under conditions of highstringency.

Just as PSA polypeptide fragments and polypeptide variants are employedby skilled artisans for use in methods of monitoring this molecule,20P2H8 polypeptide fragments and polypeptide variants can also be usedin an analogous manner. In particular, typical PSA polypeptides used inmethods of monitoring this molecule are fragments of the PSA proteinwhich contain an epitope that can be recognized by an antibody whichwill specifically bind to the PSA protein. This practice of usingpolypeptide fragments or polypeptide variants used to generateantibodies (such as anti-PSA antibodies) is typical in the art with awide variety of systems such as fusion proteins being used bypractitioners (see e.g. Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995). In this context,each of the variety of epitopes in a protein of interest functions toprovide the architecture upon which the antibody is generated.Typically, skilled artisans generally create a variety of differentpolypeptide fragments that can be used in order to generate antibodiesspecific for different portions of a polypeptide of interest (see e.g.U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 20P2H8biological motifs discussed below. Polypeptide fragments and variantsare typically useful in this context as long as they have the commonattribute or characteristic of having an epitope capable of generatingan antibody specific for a target polypeptide sequence (e.g. the 20P2H8polypeptide shown in SEQ ID NO: 2).

As shown herein, the 20P2H8 polynucleotides and polypeptides (as well asthe 20P2H8 polynucleotide probes and anti-20P2H8 antibodies used toidentify the presence of these molecules) exhibit specific propertiesthat make them useful in diagnosing cancers of the prostate. Thedescribed diagnostic assays that measures the presence of 20P2H8 geneproducts, in order to evaluate the presence or onset of the particulardisease conditions described herein such as prostate cancer areparticularly useful in identifying potential candidates for preventivemeasures or further monitoring, as has been done so successfully withPSA. Moreover, these materials satisfy a need in the art for moleculeshaving similar characteristics to PSA in situations where, for example,a definite diagnosis of metastasis of prostatic origin cannot be made onthe basis of a testing for PSA alone (see e.g. Alanen et al., Pathol.Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as20P2H8 polynucleotides and polypeptides (as well as the 20P2H8polynucleotide probes and anti-20P2H8 antibodies used to identify thepresence of these molecules) must be employed to confirm metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 20P2H8polynucleotides disclosed herein have a number of other specificutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in 15q22. Moreover, in addition totheir use in diagnostic assays, the 20P2H8 polypeptides andpolynucleotides disclosed herein have other utilities such as their usein the forensic analysis of tissues of unknown origin (see e.g. TakahamaK Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

20P2H8 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 20P2H8 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 20P2H8 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a 20P2H8 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides that hybridize to a 20P2H8 gene,mRNA, or to a 20P2H8 encoding polynucleotide (collectively, “20P2H8polynucleotides”). As used herein, the 20P2H8 gene and protein is meantto include the 20P2H8 genes and proteins specifically described hereinand the genes and proteins corresponding to other 20P2H8 proteins andstructurally similar variants of the foregoing. Such other 20P2H8proteins and variants will generally have coding sequences that arehighly homologous to the 20P2H8 coding sequence, and preferably willshare at least about 50% amino acid identity and at least about 60%amino acid homology (using BLAST criteria), more preferably sharing 70%or greater homology (using BLAST criteria).

A 20P2H8 polynucleotide may comprise a polynucleotide having thenucleotide sequence of human 20P2H8 as shown in FIG. 1, wherein T canalso be U; a polynucleotide which encodes all or part of the 20P2H8protein; a sequence complementary to the foregoing; or a polynucleotidefragment of any of the foregoing. Another embodiment comprises apolynucleotide encoding a 20P2H8 polypeptide whose sequence is encodedby the cDNA contained in the plasmid as deposited with American TypeCulture Collection 10801 University Boulevard, Manassas, Va., USA (ATCC)as Accession No. 207151. Another embodiment comprises a polynucleotidewhich is capable of hybridizing under stringent hybridization conditionsto the human 20P2H8 cDNA shown in FIG. 1.

Typical embodiments of the invention disclosed herein include 20P2H8polynucleotides containing specific portions of the 20P2H8 mRNA sequence(and those which are complementary to such sequences) such as those thatencode the protein and fragments thereof. For example, representativeembodiments of the invention disclosed herein include: polynucleotidesencoding about amino acid 1 to about amino acid 10 of the 20P2H8 proteinshown in SEQ ID NO: 2, polynucleotides encoding about amino acid 20 toabout amino acid 30 of the 20P2H8 protein shown in SEQ ID NO: 2,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 20P2H8 protein shown in SEQ ID NO: 2, polynucleotides encoding aboutamino acid 40 to about amino acid 50 of the 20P2H8 protein shown in SEQID NO: 2, polynucleotides encoding about amino acid 50 to about aminoacid 60 of the 20P2H8 protein shown in SEQ ID NO: 2, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the 20P2H8protein shown in SEQ ID NO: 2, polynucleotides encoding about amino acid70 to about amino acid 80 of the 20P2H8 protein shown in SEQ ID NO: 2,polynucleotides encoding about amino acid 80 to about amino acid 90 ofthe 20P2H8 protein shown in SEQ ID NO: 2 and polynucleotides encodingabout amino acid 90 to about amino acid 100 of the 20P2H8 protein shownin SEQ ID NO: 2, etc. Following this scheme, polynucleotides encodingportions of the amino acid sequence of amino acids 100-517 of the 20P2H8protein (SEQ ID NO: 2) are typical embodiments of the invention.Polynucleotides encoding larger portions of the 20P2H8 protein are alsocontemplated. For example polynucleotides encoding from about amino acid1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50etc.) of the 20P2H8 protein shown in SEQ ID NO: 2 may be generated by avariety of techniques well known in the art.

Additional illustrative embodiments of 20P2H8 polynucleotides includeembodiments consisting of a polynucleotide having the sequence as shownin FIG. 1 (SEQ ID NO: 1) from about nucleotide residue number 1 throughabout nucleotide residue number 500, from about nucleotide residuenumber 500 through about nucleotide residue number 1000 and from aboutnucleotide residue number 1000 through about nucleotide residue number3600. These polynucleotide fragments can include any portion of the20P2H8 sequence as shown in FIG. 1 (SEQ ID NO: 1), for example apolynucleotide having the 517 amino acid ORF within the polynucleotidesequence as shown in FIG. 1 (SEQ ID NO: 1), e.g. from about nucleotideresidue number 450 through about nucleotide residue number 2000.Alternatively, a polynucleotide may include portions of both the codingand non-coding regions of the 20P2H8 protein such as a polynucleotidefragment consisting of the sequence from about residue 600 through aboutresidue 3600 etc.

Additional illustrative embodiments of the invention disclosed hereininclude 20P2H8 polynucleotide fragments encoding one or more of thebiological motifs contained within the 20P2H8 protein sequence. Typicalpolynucleotide fragments of the invention include those that encode oneor more of the 20P2H₈N-glycosylation sites, casein kinase IIphosphorylation sites, the RNA binding sequences such as theribonucleoprotein-1 and ribonucleoprotein-2 consensus sites, the prolinerich regions or N-myristoylation sites as disclosed in greater detail inthe text discussing the 20P2H8 protein and polypeptides below.

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. For example, because the human 20P2H8 gene mapsto chromosome 15q22.32-23, polynucleotides encoding different regions ofthe 20P2H8 protein can be used to characterize cytogenetic abnormalitieson chromosome 15, bands q13.2-q14 that have been identified as beingassociated with various cancers. In particular, a variety of chromosomalabnormalities in 15q22.32-23 have been identified as frequentcytogenetic abnormalities in a number of different cancers (see, e.g.,Goffman et al., Cancer Genet. Cytogenet. 1983 8(3): 197-202; Yeatment etal., Clin. Exp. Metastasis 1996 14(3): 246-252)). Consequently,polynucleotides encoding specific regions of the 20P2H8 protein providenew tools that can be used to delineate with a greater precision thanpreviously possible, the specific nature of the cytogeneticabnormalities in this region of chromosome 15 that may contribute to themalignant phenotype. In this context, these polynucleotides satisfy aneed in the art for expanding the sensitivity of chromosomal screeningin order to identify more subtle and less common chromosomalabnormalities (see, e.g., Evans et al., 1994, Am. J. Obstet. Gynecol.171(4):1055-1057).

Alternatively, as 20P2H8 is shown to be highly expressed in prostatecancers (see e.g. FIG. 2), these polynucleotides may be used in methodsassessing the status of 20P2H8 gene products in normal versus canceroustissues. Typically, polynucleotides encoding specific regions of the20P2H8 protein may be used to assess the presence of perturbations (suchas deletions, insertions, point mutations etc.) in specific regions(such as regions containing the RNA binding sequences) of the 20P2H8gene products. Exemplary assays include both RT-PCR assays as well assingle-strand conformation polymorphism (SSCP) analysis (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8): 369-378), both of whichutilize polynucleotides encoding specific regions of a protein toexamine these regions within the protein.

Other specifically contemplated embodiments of the invention disclosedherein are genomic DNA, cDNAs, ribozymes, and antisense molecules, aswell as nucleic acid molecules based on an alternative backbone orincluding alternative bases, whether derived from natural sources orsynthesized. For example, antisense molecules can be RNAs or othermolecules, including peptide nucleic acids (PNAs) or non-nucleic acidmolecules such as phosphorothioate derivatives, that specifically bindDNA or RNA in a base pair-dependent manner. A skilled artisan canreadily obtain these classes of nucleic acid molecules using the 20P2H8polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,20P2H8. See for example, Jack Cohen, 1988, OLIGODEOXYUCLEOTIDES,Antisense Inhibitors of Gene Expression, CRC Press; and Synthesis 1:1-5(1988). The 20P2H8 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention may beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P. et al, 1990, J. Org. Chem. 55:4693-4698; andIyer, R. P. et al., 1990, J. Am. Chem. Soc. 112:1253-1254, thedisclosures of which are fully incorporated by reference herein.Additional 20P2H8 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see e.g.Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6:169-175).

The 20P2H8 antisense oligonucleotides of the present invention typicallymay be RNA or DNA that is complementary to and stably hybridizes withthe first 100 N-terminal codons or last 100 C-terminal codons of the20P2H8 genomic sequence or the corresponding mRNA. While absolutecomplementarity is not required, high degrees of complementatity arepreferred. Use of an oligonucleotide complementary to this region allowsfor the selective hybridization to 20P2H8 mRNA and not to mRNAspecifying other regulatory subunits of protein kinase. Preferably, the20P2H8 antisense oligonucleotides of the present invention are a 15 to30-mer fragment of the antisense DNA molecule having a sequence thathybridizes to 20P2H8 mRNA. Optionally, 20P2H8 antisense oligonucleotideis a 30-mer oligonucleotide that is complementary to a region in thefirst 10 N-terminal codons and last 10 C-terminal codons of 20P2H8.Alternatively, the antisense molecules are modified to employ ribozymesin the inhibition of 20P2H8 expression (L. A. Couture & D. T.Stinchcomb, 1996, Trends Genet. 12: 510-515).

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a 20P2H8 polynucleotide in a sample and as ameans for detecting a cell expressing a 20P2H8 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 20P2H8 cDNA sequences shown in SEQ ID NO: 1. Examples ofprimer pairs capable of specifically amplifying 20P2H8 mRNAs are alsodescribed in the Examples that follow. As will be understood by theskilled artisan, a great many different primers and probes may beprepared based on the sequences provided herein and used effectively toamplify and/or detect a 20P2H8 mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the 20P2H8 gene or that encodepolypeptides other than 20P2H8 gene product or fragments thereof. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated 20P2H8 polynucleotide.

The 20P2H8 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 20P2H8 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 20P2H8 polypeptides; as tools for modulatingor inhibiting the expression of the 20P2H8 gene(s) and/or translation ofthe 20P2H8 transcript(s); and as therapeutic agents.

Isolation of 20P2H8-Encoding Nucleic Acid Molecules

The 20P2H8 cDNA sequences described herein enable the isolation of otherpolynucleotides encoding 20P2H8 gene product(s), as well as theisolation of polynucleotides encoding 20P2H8 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 20P2H8 gene product. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 20P2H8 gene are wellknown (See, e.g., Sambrook, J. et al., 1989, Molecular Cloning: ALaboratory Manual 2d ed., Cold Spring Harbor Press, New York; Ausubel etal., eds., 1995, Current Protocols in Molecular Biology, Wiley andSons). For example, lambda phage cloning methodologies may beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 20P2H8gene cDNAs may be identified by probing with a labeled 20P2H8 cDNA or afragment thereof. For example, in one embodiment, the 20P2H8 cDNA (SEQID NO: 1) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full length cDNAs corresponding to a 20P2H8gene. The 20P2H8 gene itself may be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 20P2H8 DNAprobes or primers.

Recombinant DNA Molecules And Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 20P2H8 polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, e.g., Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 20P2H8 polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovitus-infectible cell such as an Sf9 or HighFive cell). Examplesof suitable mammalian cells include various prostate cancer cell linessuch as PrEC, LNCaP and TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of 20P2H8 may be used to generate 20P2H8 proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of20P2H8 proteins or fragments thereof are available (see, e.g., Sambrooket al., 1989, supra; Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expressionvectors, 20P2H8 may be preferably expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 20P2H8 protein or fragment thereof. Such host-vectorsystems may be employed to study the functional properties of 20P2H8 and20P2H8 mutations.

Recombinant human 20P2H8 protein may be produced by mammalian cellstransfected with a construct encoding 20P2H8. In an illustrativeembodiment described in the Examples, 293T cells can be transfected withan expression plasmid encoding 20P2H8, the 20P2H8 protein is expressedin the 293T cells, and the recombinant 20P2H8 protein can be isolatedusing standard purification methods (e.g., affinity purification usinganti-20P2H8 antibodies). In another embodiment, also described in theExamples herein, the 20P2H8 coding sequence is subcloned into theretroviral vector pSRαMSVtkneo and used to infect various mammalian celllines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish20P2H8 expressing cell lines. Various other expression systems wellknown in the art may also be employed. Expression constructs encoding aleader peptide joined in frame to the 20P2H8 coding sequence may be usedfor the generation of a secreted form of recombinant 20P2H8 protein.

Proteins encoded by the 20P2H8 genes, or by fragments thereof, will havea variety of uses, including but not limited to generating antibodiesand in methods for identifying ligands and other agents and cellularconstituents that bind to a 20P2H8 gene product. Antibodies raisedagainst a 20P2H8 protein like 20P2H8 polynucleotides) or fragmentthereof may be useful in diagnostic and prognostic assays, and imagingmethodologies in the management of human cancers characterized byexpression of 20P2H8 protein, including but not limited to cancers ofthe kidney, skin, cervix, prostate, brain, bladder, pancreas, ovaries,lung, testis and breast (see e.g. FIGS. 4-8). Such antibodies may beexpressed intracellularly and used in methods of treating patients withsuch cancers. Various immunological assays useful for the detection of20P2H8 proteins are contemplated, including but not limited to varioustypes of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting 20P2H8 expressingcells (e.g., in radioscintigraphic imaging methods). 20P2H8 proteins mayalso be particularly useful in generating cancer vaccines, as furtherdescribed below.

20P2H8 Polypeptides

Another aspect of the present invention provides 20P2H8 proteins andpolypeptide fragments thereof. The 20P2H8 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined below. Fusion proteins that combine partsof different 20P2H8 proteins or fragments thereof, as well as fusionproteins of a 20P2H8 protein and a heterologous polypeptide are alsoincluded. Such 20P2H8 proteins will be collectively referred to as the20P2H8 proteins, the proteins of the invention, or 20P2H8. As usedherein, the term “20P2H8 polypeptide” refers to a polypeptide fragmentor a 20P2H8 protein of at least 6 amino acids, preferably at least 15amino acids.

Specific embodiments of 20P2H8 proteins comprise a polypeptide havingthe amino acid sequence of human 20P2H8 as shown in SEQ ID NO: 2.Alternatively, embodiments of 20P2H8 proteins comprise variantpolypeptides having alterations in the amino acid sequence of human20P2H8 as shown in SEQ ID NO: 2.

In general, naturally occurring allelic variants of human 20P2H8 willshare a high degree of structural identity and homology (e.g., 90% ormote identity). Typically, allelic variants of the 20P2H8 proteins willcontain conservative amino acid substitutions within the 20P2H8sequences described herein or will contain a substitution of an aminoacid from a corresponding position in a 20P2H8 homologue. One class of20P2H8 allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular 20P2H8 amino acidsequence, but will farther contain a radical departure from thesequence, such as a non-conservative substitution, truncation, insertionor frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (O)for asparagine (N) and vice versa; and serine (S) for threonine (I) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

Embodiments of the invention disclosed herein include a wide variety ofart accepted variants of 20P2H8 proteins such as polypeptides havingamino acid insertions, deletions and substitutions. 20P2H8 variants canbe made using methods known in the art such as site-directedmutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis (Carter et al., 1986, Nucl. Acids Res. 13:4331; Zoller etal., 1987, Nucl. Acids Res. 10:6487), cassette mutagenesis (Wells etal., 1985, Gene 34:315), restriction selection mutagenesis (Wells etal., 1986, Philos. Trans. R. Soc. London Ser. A, 317:415) or other knowntechniques can be performed on the cloned DNA to produce the 20P2H8variant DNA. Scanning amino acid analysis can also be employed toidentify one or more amino acids along a contiguous sequence. Among thepreferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is typically a preferred scanning amino acid among this groupbecause it eliminates the side-chain beyond the beta-carbon and is lesslikely to alter the main-chain conformation of the variant. Alanine isalso typically preferred because it is the most common amino acid.Further, it is frequently found in both buried and exposed positions(Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, 1976, J.Mol. Biol., 150:1). If alanine substitution does not yield adequateamounts of variant, an isosteric amino acid can be used.

As defined herein, 20P2H8 variants have the distinguishing attribute ofhaving at least one epitope in common with a 20P2H8 protein having theamino acid sequence of SEQ ID NO: 2, such that an antibody thatspecifically binds to a 20P2H8 variant will also specifically bind tothe 20P2H8 protein having the amino acid sequence of SEQ ID NO: 2. Apolypeptide ceases to be a variant of the protein shown in SEQ ID NO: 2when it no longer contains an epitope capable of being recognized by anantibody that specifically binds to a 20P2H8 protein. Those skilled inthe art understand that antibodies that recognize proteins bind toepitopes of varying size, and a grouping of the order of about six aminoacids, contiguous or not, is regarded as a typical number of amino acidsin a minimal epitope. See e.g. Hebbes et al., Mol Immunol (1989)26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608. Asthere are approximately 20 amino acids that can be included at a givenposition within the minimal 6 amino acid epitope, an approximation ofthe odds of such an epitope occurring by chance are about 20⁶ or about 1in 64 million. Another specific class of 20P2H8 protein variants shares90% or more identity with the amino acid sequence of SEQ ID NO: 2.Another specific class of 20P2H8 protein variants comprises one or moreof the 20P2H8 biological motifs described below.

As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 517 amino acid sequence of the20P2H8 protein shown in SEQ ID NO: 2 (and the polynucleotides encodingsuch polypeptides). For example, representative embodiments of theinvention disclosed herein include polypeptides consisting of aboutamino acid 1 to about amino acid 10 of the 20P2H8 protein shown in SEQID NO: 2, polypeptides consisting of about amino acid 20 to about aminoacid 30 of the 20P2H8 protein shown in SEQ ID NO: 2, polypeptidesconsisting of about amino acid 30 to about amino acid 40 of the 20P2H8protein shown in SEQ ID NO: 2, polypeptides consisting of about aminoacid 40 to about amino acid 50 of the 20P2H8 protein shown in SEQ ID NO:2, polypeptides consisting of about amino acid 50 to about amino acid 60of the 20P2H8 protein shown in SEQ ID NO: 2, polypeptides consisting ofabout amino acid 60 to about amino acid 70 of the 20P2H8 protein shownin SEQ ID NO: 2, polypeptides consisting of about amino acid 70 to aboutamino acid 80 of the 20P2H8 protein shown in SEQ ID NO: 2, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 20P2H8protein shown in SEQ ID NO: 2 and polypeptides consisting of about aminoacid 90 to about amino acid 100 of the 20P2H8 protein shown in SEQ IDNO: 2, etc. Following this scheme, polypeptides consisting of portionsof the amino acid sequence of amino acids 100-517 of the 20P2H8 proteinare typical embodiments of the invention. Polypeptides consisting oflarger portions of the 20P2H8 protein are also contemplated. For examplepolypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 30, or 40 or 50 etc.) of the 20P2H8 proteinshown in SEQ ID NO: 2 may be generated by a variety of techniques wellknown in the art.

Additional illustrative embodiments of the invention disclosed hereininclude 20P2H8 polypeptides containing the amino acid residues of one ormore of the biological motifs contained within the 20P2H8 polypeptidesequence as shown in SEQ ID NO: 2. In one embodiment, typicalpolypeptides of the invention can contain one or more of the20P2H₈N-glycosylation sites such as NYTA at residues 436-439 and/or NLSGat residues 459-462 (SEQ ID NO: 2). In another embodiment, typicalpolypeptides of the invention can contain one or more of the 20P2H8casein kinase II phosphorylation sites such as SQVE at residues 19-22,SDPE at residues 41-44, SKME at residues 56-59, SDQD at residues 77-80,TGED at residues 141-144, TSNE at residues 152-155, TAEE at residues178-181, TYPD at residues 203-206, TAAE at residues 245-248, TIED atresidues 297-300 and/or SAEE at residues 364-367 (SEQ ID NO: 2). Inanother embodiment, typical polypeptides of the invention can containone or more of the N-myristoylation sites such as GLNIAK at residues87-92, GAALCL at residues 94-99, GGTSNE at residues 150-155, GLPFTA atresidues 172-177, GGKEGI at residues 194-199, GLPYAA at residues291-296, GGTLNR at residues 374-379, GSPNSL at residues 446-451, GLAYNTat residues 475-480 and/or GLIHTN at residues 499-504 (SEQ ID NO: 2). Inanother embodiment, typical polypeptides of the invention can containthe one or more of the amidation sites such as QGRR at residues 102-105and/or LGKR at residues 234-237 (SEQ ID NO: 2). In another embodiment,typical polypeptides of the invention can contain one or more of theRNA-binding domains such as those shown in FIG. 1. In anotherembodiment, typical polypeptides of the invention can contain one ormore of the proline rich regions such as those shown in FIG. 1. Inanother embodiment, typical polypeptides of the invention can containone or more of the immunoreactive epitopes identified by a processdescribed herein such as such as those shown in. Table 1. Relatedembodiments of these inventions include polypeptides containingcombinations of the different motifs discussed above with preferableembodiments being those which contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthese polypeptides. In addition, embodiments which include a number ofeither N-terminal and/or C-terminal amino acid residues on either sideof these motifs may be desirable (to, for example, include a greaterportion of the polypeptide architecture in which the motif is located).Typically the number of N-terminal and/or C-terminal amino acid residueson either side of a motif is between about 5 to about 50 amino acidresidues.

Illustrative examples of such embodiments includes a polypeptide havingone or more motifs selected from the group consisting of SDPE and/orSKME and/or TGED (SEQ ID NO: 2). Alternatively polypeptides having othercombinations of the biological motifs disclosed herein ate alsocontemplated such as a polypeptide having GLIHN and any one of the otherbiological motifs such as SKME or a polypeptide having GLIHN and any oneof the other biological motifs such as GGKEGI etc. (SEQ ID NO: 2).

Polypeptides consisting of one or more of the 20P2H8 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 20P2H8 motifsdiscussed above are associated with growth dysregulation and because20P2H8 is overexpressed in cancers (FIGS. 4 and 5). Casein kinase II andprotein kinase C for example are enzymes known to be associated with thedevelopment of the malignant phenotype (see e.g. Chen et al., 1998, LabInvest, 78(2):165-174; Gaiddon et al., 1995, Endocrinology136(10):4331-4338; Hall et al., 1996, Nucleic Acids Research24(6):1119-1126; Peterziel et al., 1999, Oncogene 18(46):6322-6329; andO'Brian, 1998, Oncol. Rep. 5(2): 305-309). Moreover, both glycosylationand myristoylation are protein modifications also associated with cancerand cancer progression (see e.g. Dennis et al., 1999, Biochim. Biophys.Acta 1473(1):21-34; Raju et al., 1997, Exp. Cell Res. 235(1):145-154).In addition, the RNA recognition motifs and proline rich regions arealso associated with oncogenic processes (see e.g. Kennedy et al. Nat.Genet. 12(3): 329-331 (1996): Li et al., J. Biol. Chem. 275(30):23053-23058 (2000) and Xiao et al., Blood. 2000 Jul. 15;96(2):699-704).

The polypeptides of the preceding paragraphs have a number of differentspecific uses. As 20P2H8 is shown to be expressed in a variety ofcancers including kidney, prostate, bladder, testicular, ovarian,breast, pancreas, colon, skin, cervical, stomach and lung cancer celllines and/or patient samples (see e.g. FIGS. 4-8), these polypeptidesmay be used in methods assessing the status of 20P2H8 gene products innormal versus cancerous tissues and elucidating the malignant phenotype.Typically, polypeptides encoding specific regions of the 20P2H8 proteinmay be used to assess the presence of perturbations (such as deletions,insertions, point mutations etc.) in specific regions of the 20P2H8 geneproducts (such as regions containing the RNA binding motifs). Exemplaryassays can utilize antibodies targeting a 20P2H8 polypeptide containingthe amino acid residues of one or more of the biological motifscontained within the 20P2H8 polypeptide sequence in order to evaluatethe characteristics of this region in normal versus cancerous tissues.Alternatively, 20P2H8 polypeptides containing the amino acid residues ofone or more of the biological motifs contained within the 20P2H8polypeptide sequence can be used to screen for factors that interactwith that region of 20P2H8.

As discussed above, redundancy in the genetic code permits variation in20P2H8 gene sequences. In particular, one skilled in the art willrecognize specific codon preferences by a specific host species and canadapt the disclosed sequence as preferred for a desired host. Forexample, preferred codon sequences typically have rare codons (i.e.,codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific organism may be calculated, forexample, by utilizing codon usage tables available on the Internet atthe following address: http://www.dna.affrc.go.jp/˜nakamura/codon.html.Nucleotide sequences that have been optimized for a particular hostspecies by replacing any codons having a usage frequency of less thanabout 20% are referred to herein as “codon optimized sequences.”

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that may be deleterious to gene expression. The GC content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. Where possible, the sequence may also be modified to avoidpredicted hairpin secondary mRNA structures. Other useful modificationsinclude the addition of a translational initiation consensus sequence atthe start of the open reading frame, as described in Kozak, 1989, Mol.Cell Biol., 9:5073-5080. Nucleotide sequences that have been optimizedfor expression in a given host species by elimination of spuriouspolyadenylation sequences, elimination of exon/intron splicing signals,elimination of transposon-like repeats and/or optimization of GC contentin addition to codon optimization are referred to herein as an“expression enhanced sequence.”

20P2H8 proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 20P2H8 proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 20P2H8 protein. A purified 20P2H8 proteinmolecule will be substantially free of other proteins or molecules thatimpair the binding of 20P2H8 to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 20P2H8 protein include a purified 20P2H8 protein and afunctional, soluble 20P2H8 protein. In one form, such functional,soluble 20P2H8 proteins or fragments thereof retain the ability to bindantibody or other ligand.

The invention also provides 20P2H8 polypeptides comprising biologicallyactive fragments of the 20P2H8 amino acid sequence, such as apolypeptide corresponding to part of the amino acid sequence for 20P2H8as shown in SEQ ID NO: 2. Such polypeptides of the invention exhibitproperties of the 20P2H8 protein, such as the ability to elicit thegeneration of antibodies that specifically bind an epitope associatedwith the 20P2H8 protein.

20P2H8 polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human 20P2H8 proteins disclosedherein. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a polypeptide fragment of a 20P2H8protein. In this regard, the 20P2H8-encoding nucleic acid moleculesdescribed herein provide means for generating defined fragments of20P2H8 proteins. 20P2H8 polypeptides are particularly useful ingenerating and characterizing domain specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a20P2H8 protein), in identifying agents or cellular factors that bind to20P2H8 or a particular structural domain thereof, and in varioustherapeutic contexts, including but not limited to cancer vaccines.

20P2H8 polypeptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-20P2H8 antibodies or in identifying cellular factors thatbind to 20P2H8.

In an embodiment described in the examples that follow, 20P2H8 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 20P2H8 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 20P2H8 protein intransfected cells. The secreted HIS-tagged 20P2H8 in the culture mediamay be purified using a nickel column using standard techniques.

Modifications of 20P2H8 such as covalent modifications are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a 20P2H8 polypeptidewith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the 20P2H8.Another type of covalent modification of the 20P2H8 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence 20P2H8(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequence20P2H8. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present. Anothertype of covalent modification of 20P2H8 comprises linking the 20P2H8polypeptide to one of a variety of nonproteinacceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The 20P2H8 of the present invention may also be modified in a way toform a chimeric molecule comprising 20P2H8 fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of the 20P2H8 with apolyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the 20P2H8. In analternative embodiment, the chimeric molecule may comprise a fusion ofthe 20P2H8 with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 20P2H8 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

20P2H8 Antibodies

The term “antibody” is used in the broadest sense and specificallycovers single anti-20P2H8 monoclonal antibodies (including agonist,antagonist and neutralizing antibodies) and anti-20P2H8 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” (mAb) as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiescomprising the individual population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

Another aspect of the invention provides antibodies that bind to 20P2H8proteins and polypeptides. The most preferred antibodies willspecifically bind to a 20P2H8 protein and will not bind (or will bindweakly) to non-20P2H8 proteins and polypeptides. Anti-20P2H8 antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule thatbinds to its target, i.e., the antigen binding region.

20P2H8 antibodies of the invention may be particularly useful inprostate cancer diagnostic and prognostic assays, and imagingmethodologies. Intracellularly expressed antibodies (e.g., single chainantibodies) may be therapeutically useful in treating cancers in whichthe expression of 20P2H8 is involved, such as for example advanced andmetastatic prostate cancers. Such antibodies may be useful in thetreatment, diagnosis, and/or prognosis of other cancers, to the extent20P2H8 is also expressed or overexpressed in other types of cancers suchas prostate, kidney, bladder, cervical, skin, stomach, testicular,ovarian, breast, pancreas, colon and lung cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 20P2H8 and mutant 20P2H8 proteins andpolypeptides. Such assays generally comprise one or more 20P2H8antibodies capable of recognizing and binding a 20P2H8 or mutant 20P2H8protein, as appropriate, and may be performed within variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer and other cancers expressing 20P2H8are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled 20P2H8 antibodies. Suchassays may be clinically useful in the detection, monitoring, andprognosis of 20P2H8 expressing cancers such as prostate, bladder,testicular, ovarian, breast, pancreas, colon and lung cancer cell lines.

20P2H8 antibodies may also be used in methods for purifying 20P2H8 andmutant 20P2H8 proteins and polypeptides and for isolating 20P2H8homologues and related molecules. For example, in one embodiment, themethod of purifying a 20P2H8 protein comprises incubating a 20P2H8antibody, which has been coupled to a solid matrix, with a lysate orother solution containing 20P2H8 under conditions that permit the 20P2H8antibody to bind to 20P2H8; washing the solid matrix to eliminateimpurities; and eluting the 20P2H8 from the coupled antibody. Other usesof the 20P2H8 antibodies of the invention include generatinganti-idiotypic antibodies that mimic the 20P2H8 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a 20P2H8 protein, peptide, or fragment, in isolatedor immunoconjugated form (Harlow, and Lane, eds., 1988, Antibodies: ALaboratory Manual, CSH Press; Harlow, 1989, Antibodies, Cold SpringHarbor Press, N.Y.). In addition, fusion proteins of 20P2H8 may also beused, such as a 20P2H8 GST-fusion protein. In a particular embodiment, aGST fusion protein comprising all or most of the open reading frameamino acid sequence of SEQ ID NO: 2 may be produced and used as animmunogen to generate appropriate antibodies. In another embodiment, a20P2H8 peptide may be synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art may beused (with or without purified 20P2H8 protein or 20P2H8 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

The amino acid sequence of the 20P2H8 as shown in SEQ ID NO: 2 may beused to select specific regions of the 20P2H8 protein for generatingantibodies. For example, hydrophobicity and hydrophilicity analyses ofthe 20P2H8 amino acid sequence may be used to identify hydrophilicregions in the 20P2H8 structure. Regions of the 20P2H8 protein that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis.

Illustrating this, the binding of peptides from 20P2H8 proteins to thehuman MHC class I molecule HLA-A2 are predicted and shown in Table 1below. Specifically, the complete amino acid sequences of 20P2H8proteins was entered into the HLA Peptide Motif Search algorithm foundin the Bioinformatics and Molecular Analysis Section (BIMAS) Web site(http://bimas.dcrt.nih.gov/). The HLA Peptide Motif Search algorithm wasdeveloped by Dr. Ken Parker based on binding of specific peptidesequences in the groove of HLA Class I molecules and specifically HLA-A2(see e.g. Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parkeret al., J. Immunol. 152:163-75 (1994)). This algorithm allows locationand ranking of 8-mer, 9-mer, and 10-mer peptides from a complete proteinsequence for predicted binding to HLA-A2 as well as other HLA Class Imolecules. Most HLA-A2 binding peptides are 9-mers favorably containinga leucine (L) at position 2 and a valine (V) or leucine (L) at position9 (Parker et al., J. Immunol. 149:3580-7 (1992)). The results of 20P2H8predicted binding peptides are shown in Table 1 below. In Table 1, thetop 10 ranking candidates for each family member are shown along withtheir location, the amino acid sequence of each specific peptide, and anestimated binding score. The binding score corresponds to the estimatedhalf-time of dissociation of complexes containing the peptide at 37° C.at pH 6.5. Peptides with the highest binding score are predicted to bethe most tightly bound to HLA Class I on the cell surface and thusrepresent the best immunogenic targets for T-cell recognition. Actualbinding of peptides to HLA-A2 can be evaluated by stabilization ofHLA-A2 expression on the antigen-processing defective cell line T2 (seee.g. Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate36:129-38 (1998)). Immunogenicity of specific peptides can be evaluatedin vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in thepresence of dendritic cells.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a 20P2H8 immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

20P2H8 monoclonal antibodies are preferred and may be produced byvarious means well known in the art. For example, immortalized celllines that secrete a desired monoclonal antibody may be prepared usingthe standard hybridoma technology of Kohler and Milstein ormodifications that immortalize producing B cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the 20P2H8 protein or a20P2H8 fragment. When the appropriate immortalized cell culturesecreting the desired antibody is identified, the cells may be expandedand antibodies produced either from in vitro cultures or from ascitesfluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the 20P2H8 protein can also be produced in thecontext of chimeric or CDR grafted antibodies of multiple speciesorigin. Humanized or human 20P2H8 antibodies may also be produced andare preferred for use in therapeutic contexts. Methods for humanizingmurine and other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al, 1986, Nature 321:522-525;Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988,Science 239:1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad.Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151:2296. Methodsfor producing fully human monoclonal antibodies include phage displayand transgenic methods (for review, see Vaughan et al., 1998, NatureBiotechnology 16:535-539).

Fully human 20P2H8 monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In: Clark,M., ed., 1993, Protein Engineering of Antibody Molecules forProphylactic and Therapeutic Applications in Man, Nottingham Academic,pp 45-64; Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human 20P2H8 monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest Drugs 7(4):607-614). Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 20P2H8 antibodies with a 20P2H8 protein may be establishedby a number of well known means, including western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,20P2H8 proteins, peptides, 20P2H8-expressing cells or extracts thereof.

A 20P2H8 antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. A second molecule forconjugation to the 20P2H8 antibody can be selected in accordance withthe intended use. For example, for therapeutic use, the second moleculecan be a toxin or therapeutic agent Further, bi-specific antibodiesspecific for two or more 20P2H8 epitopes may be generated using methodsgenerally known in the art. Homodimeric antibodies may also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., 1993,Cancer Res. 53: 2560-2565).

20P2H8 Transgenic Animals

Nucleic acids that encode 20P2H8 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA that is integrated into the genomeof a cell from which a transgenic animal develops. In one embodiment,cDNA encoding 20P2H8 can be used to clone genomic DNA encoding 20P2H8 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells that express DNA encoding20P2H8. Methods for generating transgenic animals, particularly animalssuch as mice or rats, have become conventional in the art and aredescribed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for 20P2H8 transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding 20P2H8 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding 20P2H8. Such animals canbe used as tester animals for reagents thought to confer protectionfrom, for example, pathological conditions associated with itsoverexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of 20P2H8 can be used to construct a20P2H8 “knock out” animal that has a defective or altered gene encoding20P2H8 as a result of homologous recombination between the endogenousgene encoding 20P2H8 and altered genomic DNA encoding 20P2H8 introducedinto an embryonic cell of the animal. For example, cDNA encoding 20P2H8can be used to clone genomic DNA encoding 20P2H8 in accordance withestablished techniques. A portion of the genomic DNA encoding 20P2H8 canbe deleted or replaced with another gene, such as a gene encoding aselectable marker that can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector (see e.g., Thomas and Capecchi, 1987, Cell51:503) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., L et al.,1992, Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras (see e.g., Bradley, in Robertson, ed., 1987, Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, (IRL, Oxford), pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the 20P2H8 polypeptide.

Methods for the Detection of 20P2H8

Another aspect of the present invention relates to methods for detecting20P2H8 polynucleotides and 20P2H8 proteins and variants thereof, as wellas methods for identifying a cell that expresses 20P2H8. The expressionprofile of 20P2H8 makes it a potential diagnostic marker for localand/or metastasized disease. Northern blot analysis suggests thatdifferent tissues express different isoforms of 20P2H8. The 20P2H8isoforms in prostate cancer appear to be different from the isoformexpressed in normal prostate. In this context, the status of 20P2H8 geneproducts may provide information useful for predicting a variety offactors including susceptibility to advanced stage disease, rate ofprogression, and/or tumor aggressiveness. As discussed in detail below,the status of 20P2H8 gene products in patient samples may be analyzed bya variety protocols that are well known in the art includingimmunohistochemical analysis, the variety of Northern blottingtechniques including in situ hybridization, RT-PCR analysis (for exampleon laser capture micro-dissected samples), western blot analysis andtissue array analysis.

More particularly, the invention provides assays for the detection of20P2H8 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 20P2HB polynucleotides include, for example, a 20P2H8gene or fragments thereof, 20P2H8 mRNA, alternative splice variant20P2H8 mRNAs, and recombinant DNA or RNA molecules containing a 20P2H8polynucleotide. A number of methods for amplifying and/or detecting thepresence of 20P2H8 polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 20P2H8 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a20P2H8 polynucleotides as sense and antisense primers to amplify 20P2H8cDNAs therein; and detecting the presence of the amplified 20P2H8 cDNA.Optionally, the sequence of the amplified 20P2H8 cDNA can be determined.In another embodiment, a method of detecting a 20P2H8 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 20P2H8 polynucleotides assense and antisense primers to amplify the 20P2H8 gene therein; anddetecting the presence of the amplified 20P2H8 gene. Any number ofappropriate sense and antisense probe combinations may be designed fromthe nucleotide sequences provided for the 20P2H8 (SEQ ID NO: 1) and usedfor this purpose.

The invention also provides assays for detecting the presence of a20P2H8 protein in a tissue of other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a 20P2H8 protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western Blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof a 20P2H8 protein in a biological sample comprises first contactingthe sample with a 20P2H8 antibody, a 20P2H8-reactive fragment thereof,or a recombinant protein containing an antigen binding region of a20P2H8 antibody; and then detecting the binding of 20P2H8 protein in thesample thereto.

Methods for identifying a cell that expresses 20P2H8 are also provided.In one embodiment, an assay for identifying a cell that expresses a20P2H8 gene comprises detecting the presence of 20P2H8 mRNA in the cell.Methods for the detection of particular mRNAs in cells are well knownand include, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled 20P2H8 riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for 20P2H8, and other amplification type detection methods,such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell that expresses a 20P2H8gene comprises detecting the presence of 20P2H8 protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and may be employed for the detection of 20P2H8proteins and 20P2H8 expressing cells.

20P2H8 expression analysis may also be useful as a tool for identifyingand evaluating agents that modulate 20P2H8 gene expression. For example,20P2H8 expression is significantly upregulated in prostate cancer, andmay also be expressed in other cancers. Identification of a molecule orbiological agent that could inhibit 20P2H8 expression or over-expressionin cancer cells may be of therapeutic value. Such an agent may beidentified by using a screen that quantifies 20P2H8 expression byRT-PCR, nucleic acid hybridization or antibody binding.

Monitoring the Status of 20P2H8 and its Products

Assays that evaluate the status of the 20P2H8 gene and 20P2H8 geneproducts in an individual may provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because 20P2H8 mRNA is so highly expressed in prostate cancersas compared to normal prostate tissue, assays that evaluate the relativelevels of 20P2H8 mRNA transcripts or proteins in a biological sample maybe used to diagnose a disease associated with 20P2H8 dysregulation suchas cancer and may provide prognostic information useful in definingappropriate therapeutic options.

Because 20P2H8 is expressed, for example, in various prostate cancerxenograft tissues and cancer cell lines, and cancer patient samples, theexpression status of 20P2H8 can provide information useful fordetermining information including the presence, stage and location ofdisplasic, precancerous and cancerous cells, predicting susceptibilityto various stages of disease, and/or for gauging tumor aggressiveness.Moreover, the expression profile makes it a potential imaging reagentfor metastasized disease. Consequently, an important aspect of theinvention is directed to the various molecular prognostic and diagnosticmethods for examining the status of 20P2H8 in biological samples such asthose from individuals suffering from, or suspected of suffering from apathology characterized by dysregulated cellular growth such as cancer.

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see e.g.Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant 20P2H8expression in prostate cancers) can allow the early detection of suchaberrant cellular physiology before a pathology such as cancer hasprogressed to a stage at which therapeutic options are more limited. Insuch examinations, the status of 20P2H8 in a biological sample ofinterest (such as one suspected of having dysregulated cell growth) canbe compared, for example, to the status of 20P2H8 in a correspondingnormal sample (e.g. a sample from that individual (or alternativelyanother individual) that is not effected by a pathology, for example onenot suspected of having dysregulated cell growth) with alterations inthe status of 20P2H8 in the biological sample of interest (as comparedto the normal sample) providing evidence of dysregulated cellulargrowth. In addition to using a biological sample that is not effected bya pathology as a normal sample, one can also use a predeterminednormative value such as a predetermined normal level of mRNA expression(see e.g. Grever et al., J. Comp. Neurol. 1996 Dec. 9;376(2):306-14 andU.S. Pat. No. 5,837,501) to compare 20P2H8 in normal versus suspectsamples.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.As specifically described herein, the status of 20P2H8 can be evaluatedby a number of parameters known in the art. Typically an alteration inthe status of 20P2H8 comprises a change in the location of 20P2H8expressing cells and/or an increase in 20P2H8 mRNA and/or proteinexpression.

Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 20P2H8 expressing cells) as well as the, level, andbiological activity of expressed gene products (such as 20P2H8 mRNApolynucleotides and polypeptides). Alterations in the status of 20P2H8can be evaluated by a wide variety of methodologies well known in theart, typically those discussed below. Typically an alteration in thestatus of 20P2H8 comprises a change in the location of 20P2H8 and/or20P2H8 expressing cells and/or an increase in 20P2H8 mRNA and/or proteinexpression.

As discussed in detail herein, in order to identify a condition orphenomenon associated with dysregulated cell growth, the status of20P2H8 in a biological sample may be evaluated by a number of methodsutilized by skilled artisans including, but not limited to genomicSouthern analysis (to examine, for example perturbations in the 20P2H8gene), northerns and/or PCR analysis of 20P2H8 mRNA (to examine, forexample alterations in the polynucleotide sequences or expression levelsof 20P2H8 mRNAs), and western and/or immunohistochemical analysis (toexamine, for example alterations in polypeptide sequences, alterationsin polypeptide localization within a sample, alterations in expressionlevels of 20P2H8 proteins and/or associations of 20P2H8 proteins withpolypeptide binding partners). Detectable 20P2H8 polynucleotidesinclude, for example, a 20P2H8 gene or fragments thereof, 20P2H8 mRNA,alternative splice variants 20P2H8, mRNAs, and recombinant DNA or RNAmolecules containing a 20P2H8 polynucleotide.

The expression profile of 20P2H8 makes it a potential diagnostic markerfor local and/or metastasized disease. In particular, the status of20P2H8 may provide information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 20P2H8 status anddiagnosing cancers that express 20P2H8, such as cancers of the prostate,bladder, testis, ovaries, breast, pancreas, colon and lung. 20P2H8status in patient samples may be analyzed by a number of means wellknown in the art including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, western blot analysis of clinical samples andcell lines, and tissue array analysis. Typical protocols for evaluatingthe status of the 20P2H8 gene and gene products can be found, forexample in Ausubul et al. eds., 1995, Current Protocols In MolecularBiology, Units. 2 [Northern Blotting], 4 [Southern Blotting], 15[Immunoblotting] and 18 [PCR Analysis].

As described above, the status of 20P2H8 in a biological sample can beexamined by a number of well known procedures in the art. For example,the status of 20P2H8 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 20P2H8 expressing cells (e.g. those that express20P2H8 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth for example, when 20P2H8 expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node). Such alterations in the status of 20P2H8in a biological sample are often associated with dysregulated cellulargrowth. Specifically, one indicator of dysregulated cellular growth isthe metastases of cancer cells from an organ of origin (such as thetestis or prostate gland) to a different area of the body (such as alymph node). In this context, evidence of dysregulated cellular growthis important for example because occult lymph node metastases can bedetected in a substantial proportion of patients with prostate cancer,and such metastases are associated with known predictors of diseaseprogression (see e.g. J Urol 1995 August;154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 20P2H8 geneproducts by determining the status of 20P2H8 gene products expressed bycells in a test tissue sample from an individual suspected of having adisease associated with dysregulated cell growth (such as hyperplasia orcancer) and then comparing the status so determined to the status of20P2H8 gene products in a corresponding normal sample, the presence ofaberrant 20P2H8 gene products in the test sample relative to the normalsample providing an indication of the presence of dysregulated cellgrowth within the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 20P2H8 mRNA or protein expression in a test cellor tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 20P2H8 mRNA may, for example, beevaluated in tissue samples including but not limited to prostate,kidney, bladder, cervical, skin, stomach, testicular, ovarian, breast,pancreas, colon and lung issues (see e.g. FIGS. 4-8). The presence ofsignificant 20P2H8 expression in any of these tissues may be useful toindicate the emergence, presence and/or severity of these cancers, sincethe corresponding normal tissues do not express 20P2H8 mRNA or expressit at lower levels.

In a related embodiment, 20P2H8 status may be determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodor assay would comprise determining the level of 20P2H8 proteinexpressed by cells in a test tissue sample and comparing the level sodetermined to the level of 20P2H8 expressed in a corresponding normalsample. In one embodiment, the presence of 20P2H8 protein is evaluated,for example, using immunohistochemical methods. 20P2H8 antibodies orbinding partners capable of detecting 20P2H8 protein expression may beused in a variety of assay formats well known in the art for thispurpose.

In other related embodiments, one can evaluate the integrity 20P2H8nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like. Such embodiments areuseful because perturbations in the nucleotide and amino acid sequencesare observed in a large number of proteins associated with a growthdysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan.Pathol. 26(8):369-4378). In this context, a wide variety of assays forobserving perturbations in nucleotide and amino acid sequences are wellknown in the art. For example, the size and structure of nucleic acid oramino acid sequences of 20P2H8 gene products may be observed by theNorthern, Southern, Western, PCR and DNA sequencing protocols discussedherein. In addition, other methods for observing perturbations innucleotide and amino acid sequences such as single strand conformationpolymorphism analysis are well known in the art (see, e.g., U.S. Pat.Nos. 5,382,510 and 5,952,170).

In another embodiment, one can examine the methylation status of the20P2H8 gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536). In another example, expression ofthe LAGE-I tumor specific gene (which is not expressed in normalprostate but is expressed in 25-50% of prostate cancers) is induced bydeoxy-azacytidine in lymphoblastoid cells, suggesting that tumoralexpression is due to demethylation (Lethe et al., Int. J. Cancer 76(6):903-908 (1998)). In this context, a variety of assays for examiningmethylation status of a gene are well known in the art. For example, onecan utilize in Southern hybridization approaches methylation-sensitiverestriction enzymes which can not cleave sequences that containmethylated CpG sites in order to assess the overall methylation statusof CpG islands. In addition, MSP (methylation specific PCR) can rapidlyprofile the methylation status of all the CpG sites present in a CpGisland of a given gene. This procedure involves initial modification ofDNA by sodium bisulfite (which will convert all unmethylated cytosinesto uracil) followed by amplification using primers specific formethylated versus unmethylated DNA. Protocols involving methylationinterference can also be found for example in Current Protocols InMolecular Biology, Units 12, Frederick M. Ausubul et al. eds., 1995.

Gene amplification provides an additional method of assessing the statusof 20P2H8, a locus that maps to 15q22.32-23, a region shown to beperturbed in a variety of cancers. Gene amplification may be measured ina sample directly, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, 1980,Proc. Nat. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis),or in situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

In addition to the tissues discussed above, biopsied tissue orperipheral blood may be conveniently assayed for the presence of cancercells, including but not limited to prostate, kidney, bladder, cervical,skin, stomach, testicular, ovarian, breast, pancreas, colon and lungcancers using for example, Northern, dot blot or RT-PCR analysis todetect 20P2H8 expression (see e.g. FIGS. 4-8). The presence of RT-PCRamplifiable 20P2H8 mRNA provides an indication of the presence of thecancer. RT-PCR detection assays for tumor cells in peripheral blood arecurrently being evaluated for use in the diagnosis and management of anumber of human solid tumors. In the prostate cancer field, theseinclude RT-PCR assays for the detection of cells expressing PSA and PSM(Verkaik et al, 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J.Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem.41:1687-1688). RT-PCR assays are well known in the art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detecting20P2H8 mRNA or 20P2H8 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 20P2H8 mRNAexpression present is proportional to the degree of susceptibility. In aspecific embodiment, the presence of 20P2H8 in prostate tissue isexamined, with the presence of 20P2H8 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). In another specific embodiment, thepresence of 20P2H8 in tissue is examined, with the presence of 20P2H8 inthe sample providing an indication of cancer susceptibility (or theemergence or existence of a tumor). In a closely related embodiment, onecan evaluate the integrity 20P2H8 nucleotide and amino acid sequences ina biological sample in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in 20P2H8 geneproducts in the sample providing an indication of cancer susceptibility(or the emergence or existence of a tumor).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of 20P2H8 mRNAor 20P2H8 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 20P2H8 mRNA or 20P2H8 proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree of20P2H8 mRNA or 20P2H8 protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness. In a specificembodiment, aggressiveness of a tumor is evaluated by determining theextent to which 20P2H8 is expressed in the tumor cells, with higherexpression levels indicating more aggressive tumors. In a closelyrelated embodiment, one can evaluate the integrity of 20P2H8 nucleotideand amino acid sequences in a biological sample in order to identifyperturbations in the structure of these molecules such as insertions,deletions, substitutions and the like, with the presence of one or moreperturbations indicating more aggressive tumors.

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of 20P2H8 mRNA or20P2H8 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 20P2H8 mRNA or 20P2H8 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 20P2H8 mRNA or 20P2H8 proteinexpression in the tumor sample over time provides information on theprogression of the cancer. In a specific embodiment, the progression ofa cancer is evaluated by determining the extent to which 20P2H8expression in the tumor cells alters over time, with higher expressionlevels indicating a progression of the cancer. In a closely relatedembodiment, one can evaluate the integrity 20P2H8 nucleotide and aminoacid sequences in a biological sample in order to identify perturbationsin the structure of these molecules such as insertions, deletions,substitutions and the like, with the presence of one or moreperturbations indicating a progression of the cancer.

The above diagnostic approaches may be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof 20P2H8 gene and 20P2H8 gene products (or perturbations in 20P2H8 geneand 20P2H8 gene products) and a factor that is associated withmalignancy as a means of diagnosing and prognosticating the status of atissue sample. In this context, a wide variety of factors associatedwith malignancy may be utilized such as the expression of genesotherwise associated with malignancy (including PSA, PSCA and PSMexpression) as well as gross cytological observations (see e.g. Bockinget al., 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein, 1995, Hum.Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51;Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods forobserving a coincidence between the expression of 20P2H8 gene and 20P2H8gene products (or perturbations in 20P2H8 gene and 20P2H8 gene products)and an additional factor that is associated with malignancy are useful,for example, because the presence of a set or constellation of specificfactors that coincide provides information crucial for diagnosing andprognosticating the status of a tissue sample.

In a typical embodiment, methods for observing a coincidence between theexpression of 20P2H8 gene and 20P2H8 gene products (or perturbations in20P2H8 gene and 20P2H8 gene products) and a factor that is associatedwith malignancy entails detecting the overexpression of 20P2H8 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample, and observing a coincidence of 20P2H8 mRNAor protein and PSA mRNA or protein overexpression. In a specificembodiment, the expression of 20P2H8 and PSA mRNA in prostatc tissue isexamined. In a preferred embodiment, the coincidence of 20P2H8 and PSAmRNA overexpression in the sample provides an indication of prostatecancer, prostate cancer susceptibility or the emergence or existence ofa prostate tumor.

Methods for detecting and quantifying the expression of 20P2H8 mRNA orprotein are described herein and use of standard nucleic acid andprotein detection and quantification technologies is well known in theart. Standard methods for the detection and quantification of 20P2H8mRNA include in situ hybridization using labeled 20P2H8 riboprobes,Northern blot and related techniques using 20P2H8 polynucleotide probes,RT-PCR analysis using primers specific for 20P2H8, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR may be used to detect and quantify 20P2H8 mRNAexpression as described in the Examples that follow. Any number ofprimers capable of amplifying 20P2H8 may be used for this purpose,including but not limited to the various primer sets specificallydescribed herein. Standard methods for the detection and quantificationof protein may be used for this purpose. In a specific embodiment,polyclonal or monoclonal antibodies specifically reactive with thewild-type 20P2H8 protein may be used in an immunohistochemical assay ofbiopsied tissue.

Identifying Molecules that Interact with 20P2H8

The 20P2H8 protein sequences disclosed herein allow the skilled artisanto identify proteins, small molecules and other agents that interactwith 20P2H8 and pathways activated by 20P2H8 via any one of a variety ofart accepted protocols. For example one can utilize one of the varietyof so-called interaction trap systems (also referred to as the“two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor and direct expression of a reportergene, the expression of which is then assayed. Typical systems identifyprotein-protein interactions in vivo through reconstitution of aeukaryotic transcriptional activator and are disclosed for example inU.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

Alternatively one can identify molecules that interact with 20P2H8protein sequences by screening peptide libraries. In such methods,peptides that bind to selected receptor molecules such as 20P2H8 areidentified by screening libraries that encode a random or controlledcollection of amino acids. Peptides encoded by the libraries areexpressed as fusion proteins of bacteriophage coat proteins, andbacteriophage particles are then screened against the receptors ofinterest.

Peptides having a wide variety of uses, such as therapeutic ordiagnostic reagents, may thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with 20P2H8 protein sequencesare disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731.

Alternatively, cell lines expressing 20P2H8 can be used to identifyprotein-protein interactions mediated by 20P2H8. This possibility can beexamined using immunoprecipitation techniques as shown by others(Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).Typically 20P2H8 protein can be immunoprecipitated from 20P2H8expressing prostate cancer cell lines using anti-20P2H8 antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express 20P2H8 (vectors mentioned above). Theimmunoprecipitated complex can be examined for protein association byprocedures such as western blotting, ³⁵S-methionine labeling ofproteins, protein microsequencing, silver staining and two dimensionalgel electrophoresis.

Small molecules that interact with 20P2H8 can be identified throughrelated embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 20P2H8's ability to mediatephosphorylation and de-phosphorylation, second messenger signaling andtumorigenesis. Typical methods are discussed for example in U.S. Pat.No. 5,928,868 and include methods for forming hybrid ligands in which atleast one ligand is a small molecule. In an illustrative embodiment, thehybrid ligand is introduced into cells that in turn contain a first anda second expression vector. Each expression vector includes DNA forexpressing a hybrid protein that encodes a target protein linked to acoding sequence for a transcriptional module. The cells further containsa reporter gene, the expression of which is conditioned on the proximityof the first and second hybrid proteins to each other, an event thatoccurs only if the hybrid ligand binds to target sites on both hybridproteins. Those cells that express the reporter gene are selected andthe unknown small molecule or the unknown hybrid protein is identified.

A typical embodiment of this invention consists of a method of screeningfor a molecule that interacts with a 20P2H8 amino acid sequence shown inFIG. 1 (SEQ ID NO: 2), comprising the steps of contacting a populationof molecules with the 20P2H8 amino acid sequence, allowing thepopulation of molecules and the 20P2H8 amino acid sequence to interactunder conditions that facilitate an interaction, determining thepresence of a molecule that interacts with the 20P2H8 amino acidsequence and then separating molecules that do not interact with the20P2H8 amino acid sequence from molecules that do interact with the20P2H8 amino acid sequence. In a specific embodiment, the method furtherincludes purifying a molecule that interacts with the 20P2H8 amino acidsequence. In a preferred embodiment, the 20P2H8 amino acid sequence iscontacted with a library of peptides.

Therapeutic Methods and Compositions

The identification of 20P2H8 as a gene that is highly expressed incancers of the prostate (and possibly other cancers), opens a number oftherapeutic approaches to the treatment of such cancers. As discussedabove, it is possible that 20P2H8 is secreted from cancer cells and inthis way modulates proliferation signals. Its potential role as atranscription factor and its high expression in prostate cancer makes ita potential target for small molecule-mediated therapy.

Accordingly, therapeutic approaches aimed at inhibiting the activity ofthe 20P2H8 protein are expected to be useful for patients suffering fromprostate cancer, testicular cancer, and other cancers expressing 20P2H8.These therapeutic approaches aimed at inhibiting the activity of the20P2H8 protein generally fall into two classes. One class comprisesvarious methods for inhibiting the binding or association of the 20P2H8protein with its binding partner or with other proteins. Another classcomprises a variety of methods for inhibiting the transcription of the20P2H8 gene or translation of 20P2H8 mRNA.

20P2H8 as a Target for Antibody-Based Therapy

The structural features of 20P2H8 indicate that this molecule is anattractive target for antibody-based therapeutic strategies. A number oftypical antibody strategies are known in the art for targeting bothextracellular and intracellular molecules (see e.g. complement and ADCCmediated killing as well as the use of intrabodies discussed below).Because 20P2H8 is expressed by cancer cells of various lineages and notby corresponding normal cells, systemic administration of20P2H8-immunoreactive compositions would be expected to exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunotherapeutic molecule tonon-target organs and tissues. Antibodies specifically reactive withdomains of 20P2H8 can be useful to treat 20P2H8expressing cancerssystemically, either as conjugates with a toxin or therapeutic agent, oras naked antibodies capable of inhibiting cell proliferation orfunction.

20P2H8 antibodies can be introduced into a patient such that theantibody binds to 20P2H8 and modulates or perturbs a function such as aninteraction with a binding partner and consequently mediates the growthinhibition and/or destruction of the cells and the tumor and/or inhibitsthe growth of the cells or the tumor. Mechanisms by which suchantibodies exert a therapeutic effect may include complement-mediatedcytolysis, antibody-dependent cellular cytotoxicity, modulating thephysiological function of 20P2H8, inhibiting ligand binding or signaltransduction pathways, modulating tumor cell differentiation, alteringtumor angiogenesis factor profiles, and/or by inducing apoptosis. 20P2H8antibodies can be conjugated to toxic or therapeutic agents and used todeliver the toxic or therapeutic agent directly to 20P2H8-bearing tumorcells. Examples of toxic agents include, but are not limited to,calchemicin, maytansinoids, radioisotopes such as ¹³¹I, ytrium, andbismuth.

Cancer immunotherapy using anti-20P2H8 antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186;Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp.), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Fortreatment of prostate cancer, for example, 20P2H8 antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation.

Although 20P2H8 antibody therapy may be useful for all stages of cancer,antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionmay be indicated for patients who have received previously one or morechemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen may be preferred for patientswho have not received chemotherapeutic treatment. Additionally, antibodytherapy may enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

It may be desirable for some cancer patients to be evaluated for thepresence and level of 20P2H8 expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative 20P2H8imaging, or other techniques capable of reliably indicating the presenceand degree of 20P2H8 expression. Immunohistochemical analysis of tumorbiopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-20P2H8 monoclonal antibodies useful in treating prostate and othercancers include those that are capable of initiating a potent immuneresponse against the tumor and those that are capable of directcytotoxicity. In this regard, anti-20P2H8 monoclonal antibodies (mAbs)may elicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, anti-20P2H8 mAbs that exert a direct biological effect ontumor growth are useful in the practice of the invention. Potentialmechanisms by which such directly cytotoxic mAbs may act includeinhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism by which a particular anti-20P2H8 mAb exerts ananti-tumor effect may be evaluated using any number of in vitro assaysdesigned to determine ADCC, ADMMC, complement-mediated cell lysis, andso forth, as is generally known in the art.

The use of murine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs may induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response may lead to the extensive formation ofimmune complexes which, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 20P2H8antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-20P2H8 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails may have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination mayexhibit synergistic therapeutic effects. In addition, the administrationof anti-20P2H8 mAbs may be combined with other therapeutic agents,including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). Theanti-20P2H8 mAbs may be administered in their “naked” or unconjugatedform, or may have therapeutic agents conjugated to them.

The anti-20P2H8 antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. Treatment will generally involve the repeatedadministration of the anti-20P2H8 antibody preparation via an acceptableroute of administration such as intravenous injection (IV), typically ata dose in the range of about 0.1 to about 10 mg/kg body weight. Doses inthe range of 10-500 mg mAb per week may be effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-20P2H8 mAb preparation may represent an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the Ab or mAbs used, the degree of 20P2H8expression in the patient, the extent of circulating shed 20P2H8antigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed 20P2H8 antigen in serum in order to assist in the determination ofthe most effective dosing regimen and related factors. Such evaluationsmay also be used for monitoring purposes throughout therapy, and may beuseful to gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Inhibition of 20P2H8 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 20P2H8 to its binding partner or ligand, or itsassociation with other protein(s) as well as methods for inhibiting20P2H8 function.

Inhibition of 20P2H8 with Intracellular Antibodies

In one approach, recombinant vectors encoding single chain antibodiesthat specifically bind to 20P2H8 may be introduced into 20P2H8expressing cells via gene transfer technologies, wherein the encodedsingle chain anti-20P2H8 antibody is expressed intracellularly, binds to20P2H8 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatmentwill be focused. This technology has been successfully applied in theart (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors. See, for example, Richardsonet al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In one embodiment, intrabodies may be used to capture 20P2H8 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals may be engineered into such 20P2H8 intrabodies inorder to achieve the desired targeting. Such 20P2H8 intrabodies may bedesigned to bind specifically to a particular 20P2H8 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 20P2H8protein may be used to prevent 20P2H8 from gaining access to thenucleus, thereby preventing it from exerting any biological activitywithin the nucleus (e.g., preventing 20P2H8 from forming transcriptioncomplexes with other factors).

In order to specifically direct the expression of such intrabodies toparticular tumor cells, the transcription of the intrabody may be placedunder the regulatory control of an appropriate tumor-specific promoterand/or enhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer may beutilized (See, for example, U.S. Pat. No. 5,919,652).

Inhibition of 20P2H8 with Recombinant Proteins

In another approach, recombinant molecules that are capable of bindingto 20P2H8 thereby preventing 20P2H8 from accessing/binding to itsbinding partner(s) or associating with other protein(s) are used toinhibit 20P2H8 function. Such recombinant molecules may, for example,contain the reactive part(s) of a 20P2H8 specific antibody molecule. Ina particular embodiment, the 20P2H8 binding domain of a 20P2H8 bindingpartner may be engineered into a dimeric fusion protein comprising two20P2H8 ligand binding domains linked to the Fc portion of a human IgG,such as human IgG1. Such IgG portion may contain, for example, theC_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1domain. Such dimeric fusion proteins may be administered in soluble formto patients suffering from a cancer associated with the expression of20P2H8, including but not limited to prostate, bladder, testicular,ovarian, breast, pancreas, colon and lung cancers, where the dimericfusion protein specifically binds to 20P2H8 thereby blocking 20P2H8interaction with a binding partner. Such dimeric fusion proteins may befurther combined into multimeric proteins using known antibody linkingtechnologies.

Inhibition of 20P2H8 Transcription or Translation

Within another class of therapeutic approaches, the invention providesvarious methods and compositions for inhibiting the transcription of the20P2H8 gene. Similarly, the invention also provides methods andcompositions for inhibiting the translation of 20P2H8 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 20P2H8gene comprises contacting the 20P2H8 gene with a 20P2H8 antisensepolynucleotide. In another approach, a method of inhibiting 20P2H8 mRNAtranslation comprises contacting the 20P2H8 mRNA with an antisensepolynucleotide. In another approach, a 20P2H8 specific ribozyme may beused to cleave the 20P2H8 message, thereby inhibiting translation. Suchantisense and ribozyme based methods may also be directed to theregulatory regions of the 20P2H8 gene, such as the 20P2H8 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a20P2H8 gene transcription factor may be used to inhibit 20P2H8 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 20P2H8 throughinterfering with 20P2H8 transcriptional activation may also be usefulfor the treatment of cancers expressing 20P2H8. Similarly, factors thatare capable of interfering with 20P2H8 processing may be useful for thetreatment of cancers expressing 20P2H8. Cancer treatment methodsutilizing such factors are also within the scope of the invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing 20P2H8(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 20P2H8 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 20P2H8 antisensepolynucleotides, ribozymes, factors capable of interfering with 20P2H8transcription, and so forth, may be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches may be combined with any one of a widevariety of chemotherapy or radiation therapy regimens. These therapeuticapproaches may also enable the use of reduced dosages of chemotherapyand/or less frequent administration, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, may beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 20P2H8 to a bindingpartner, etc.

In vivo, the effect of a 20P2H8 therapeutic composition may be evaluatedin a suitable animal model. For example, xenogenic prostate cancermodels wherein human prostate cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice, are appropriate in relation to prostatc cancer and have beendescribed (Klein et al., 1997, Nature Medicine 3:402-408). For example,PCT Patent Application WO98/16628, Sawyers et al., published Apr. 23,1998, describes various xenograft models of human prostate cancercapable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays that qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isnon-reactive with the patient's immune system. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like (see, generally, Remington's Pharmaceutical Sciences16^(th) Ed., A. Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

As noted above, the expression profile of 20P2H8 shows that it is highlyexpressed in advanced and metastasized prostate cancer. This expressionpattern is reminiscent of the Cancer-Testis (CT) antigens or MAGEs,which are testis-specific genes that are up-regulated in melanomas andother cancers (Van den Eynde and Boon, Int J Clin Lab Res. 27:81-86,1997). Due to their tissue-specific expression and high expressionlevels in cancer, the MAGEs are currently being investigated as targetsfor cancer vaccines (Durrant, Anticancer Drugs 8:727-733, 1997; Reynoldset al., Int J Cancer 72:972-976, 1997).

The invention further provides cancer vaccines comprising a 20P2H8protein or fragment thereof, as well as DNA based vaccines. In view ofthe expression of 20P2H8 cancer vaccines are expected to be effective atspecifically preventing and/or treating 20P2H8 expressing cancerswithout creating non-specific effects on non-target tissues. The use ofa tumor antigen in a vaccine for generating humoral and cell-mediatedimmunity for use in anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al, 1995, Int. J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117). Such methods can be readily practicedby employing a 20P2H8 protein, or fragment thereof, or a 20P2H8-encodingnucleic acid molecule and recombinant vectors capable of expressing andappropriately presenting the 20P2H8 immunogen. An illustrative exampleof a typical technique consists of a method of generating an immuneresponse (e.g. a humoral response) in a mammal comprising the stepsexposing the mammal's immune system to an immunoreactive epitope (e.g.an epitope of the 20P2H8 protein shown in SEQ ID NO: 2) so that themammal generates an immune response that is specific for that epitope isgenerated (e.g. antibodies that specifically recognize that epitope).

For example, viral gene delivery systems may be used to deliver a20P2H8-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8:658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a 20P2H8 protein or fragment thereof introduced into thepatient (e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human 20P2H8 cDNA may be employed. Inanother embodiment, 20P2H8 nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a 20P2H8 protein that are capable ofoptimally binding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present 20P2H8 antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al.,1996, Prostate 29:371-380). Dendritic cells can be used to present20P2H8 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed with20P2H8 peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete 20P2H8 protein.Yet another embodiment involves engineering the overexpression of the20P2H8 gene in dendritic cells using various implementing vectors knownin the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther.4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770),lentivirus, adeno-associated virus, DNA transfection (Ribas et al.,1997, Cancer Res. 57:2865-2869), and tumor-derived RNA transfection(Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressing20P2H8 may also be engineered to express immune modulators, such asGM-CSF, and used as immunizing agents.

Anti-idiotypic anti-20P2H8 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 20P2H8 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-20P2H8 antibodies that mimic an epitope ona 20P2H8 protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J. Clin. Invest 96:334-342; Herlyn et al.,1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 20P2H8. Constructs comprising DNA encoding a20P2H8 protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded 20P2H8 protein/immunogen. Expression of the 20P2H8 proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against bone, colon, pancreatic,testicular, cervical and ovarian cancers. Various prophylactic andtherapeutic genetic immunization techniques known in the art may be used(for review, see information and references published at Internetaddress www.genweb.com).

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a 20P2H8 proteinor a 20P2H8 gene or message, respectively. Where the kit utilizesnucleic acid hybridization to detect the target nucleic acid, the kitmay also have containers containing nucleotide(s) for amplification ofthe target nucleic acid sequence and/or a container comprising areporter-means, such as a biotin-binding protein, such as avidin orstreptavidin, bound to a reporter molecule, such as an enzymatic,florescent, or radioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

EXAMPLES Various aspects of the invention are further described andillustrated by way of the several examples which follow, none of whichare intended to limit the scope of the invention. Example 1

SSH-Generated Isolation of cDNA Fragment of the 20P2H8 Gene Materialsand Methods

LAPC Xenografts and Human Tissues:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408).Androgen dependent and independent LAPC-4 xenografts LAPC-4 AD and AI,respectively) and LAPC-9 AD and AI xenografts were grown in male SCIDmice and were passaged as small tissue chunks in recipient males. LAPC-4and -9 AI xenografts were derived from LAPC-4 or -9 AD tumors,respectively. Male mice bearing AD tumors were castrated and maintainedfor 2-3 months. After the tumors re-grew, the tumors were harvested andpassaged in castrated males or in female SCID mice. Human tissues forRNA and protein analyses were obtained from the Human Tissue ResourceCenter (HTRC) at the UCLA (Los Angeles, Calif.) and from QualTek, Inc.(Santa Barbara, Calif.). A benign prostatic hyperplasia tissue samplewas patient-derived.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA Synthesis Primer):

-   5′TTTTGATRCAAGCTT₃₀3′ (SEQ ID NO: 3)

Adaptor 1:

-   5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 4)-   3′GGCCCGTCCTAG5′ (SEQ ID NO: 5)

Adaptor 2:

-   5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 6)-   3′CGGCTCCTAG5′ (SEQ ID NO: 7)

PCR Primer 1:

-   5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 8)

Nested Primer (NP)1:

-   5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 9)

Nested Prier (NP)2

-   5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 10)    Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes which may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from an androgen-dependent humanprostate cancer xenograft originally derived from a prostate cancerlymph node metastasis (LAPC-4 AD) and cDNA derived from human benignhyperplasia (BPH) prostate tissue, wherein the LAPC-4 AD xenograft wasused as the source of the “tester” cDNA, and the BPH tissue was used asthe source of the “driver” cDNA.

Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)⁺ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining Dpn II digested cDNA from BPHwith a mix of digested cDNAs derived from the human cell lines HeLa,293, A431, Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50×Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18priming using the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:11) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 12) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1XPCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1×Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec, followed by a18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalP-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the 20P2H8 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs:

-   5′-TCT TGA AAC CTC CAG ACA CAA GAA-3′ (SEQ ID NO: 13)-   5′-GGA GAT GGT AGA CAC TGG TGG AGT-3′ (SEQ ID NO: 14)

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results:

The SSH experiment described in the Materials and Methods, supra, led tothe isolation of numerous candidate gene fragment clones (SSH clones).All candidate clones were sequenced and subjected to homology analysisagainst all sequences in the major public gene and EST databases inorder to provide information on the identity of the corresponding geneand to help guide the decision to analyze a particular gene fordifferential expression. In general, gene fragments which had nohomology to any known sequence in any of the searched databases, andthus considered to represent novel genes, as well as gene fragmentsshowing homology to previously sequenced expressed sequence tags (ESTs),were subjected to differential expression analysis by RT-PCR and/orNorthern analysis.

An SHH clone of 442 bp exhibiting significant homology to ESTs derivedfrom several libraries, including libraries made from fetal liver,placenta, and a pool of lung, testis and B cells was isolated anddesignated 20P2H8. Initial expression analysis by RT-PCR showed that20P2H8 is expressed in prostate and all LAPC xenografts (FIG. 2).Additionally, RT-PCR analysis of first strand cDNA derived from 16normal tissues showed expression primarily in prostate, pancreas, colonand placenta after 25 cycles of amplification (FIG. 2). Expression wasdetected in other tissues after 30 cycles of amplification (FIG. 2).This clone, therefore, was utilized for obtaining a fill length cDNAencoding 20P2H8 as described in Example 2, below.

Example 2

Isolation of Full Length cDNA Encoding the 20P2H8 gene

The isolated 20P2H8 gene fragment of 442 bp was used as a probe toidentify the full length cDNA for 20P2H8 in a human prostate cDNAlibrary. This resulted in the isolation of a 3600 bp cDNA, clone20P2H8-GTC2, which encodes a 517 amino acid open reading frame withhomology to heterogenous nuclear ribonucleoproteins (hnRNPs). Thenucleotide and deduced amino acid sequences encoded by this cDNA areshown in FIG. 1. The highest homology is with a protein identified in C.elegans (CAA92704). Significant homology is also seen with hnRNPsinvolved in mRNA splicing (ROF, ROH1 and ROH2). 20P2H8 exhibits five RNAbinding sequences, two corresponding to ribonucleoprotein-1 (RNP1)consensus sites and three corresponding to RNP2 sites (designated inFIG. 1). In addition, 20P2H8 contains three regions with significantproline content (30-42%), which lie outside regions of homology withhnRNPs (designated in FIG. 1).

Example 3

Northern Blot Analysis of 20P2H8 Gene Expression

20P2H8 mRNA expression in normal human tissues was first analyzed byNorthern blotting two multiple tissue blots obtained from Clontech (PaloAlto, Calif.), comprising a total of 16 different normal human tissues,using labeled 20P2H8 cDNA as a probe. RNA samples were quantitativelynormalized with a β-actin probe. The results are shown in FIG. 3, PanelsA and B, and indicate that, within the 16 tissues tested, the 20P2H8gene is predominantly expressed as a single 4.4 kb transcript inpancreas, with lower level expression detected in prostate and colon,and lower level expression in placenta (FIG. 3; Panels A and B). Noother normal tissues in the panel showed detectable expression.

In addition, in order to analyze 20P2H8 expression in human cancertissues and cell lines, RNAs derived from LAPC human prostate cancerxenografts and several cancer cell lines were analyzed by Northern blotusing the 20P2H8 cDNA as probe. All RNA samples were quantitativelynormalized by ethidium bromide staining and subsequent analysis with alabeled β-actin probe. The results of this analysis are presented inFIG. 3, Panel C (LAPC xenografts) and FIG. 4 (colon and pancreaticcancer cell lines). The results show up-regulated expression of 20P2H8in LAPC-9 and LAPC-4 xenografts compared to normal prostate, withhighest levels detected in LAPC-4 AD and LAPC-4 AI (FIG. 3, Panel C) aswell as high levels of expression in several pancreatic (BxPC-3, HPAC,Capan-1) and colon (CaCo-2, T84, Colo-205) cancer cell lines (FIG. 4),suggesting that 20P2H8 is a gene that is up-regulated in prostate,pancreatic and colon cancers.

Example 4

Production of Recombinant 20P2H8

To express recombinant 20P2H8 for use in a number of contexts such asanalyzing the subcellular localization of 20P2H8 protein, a partial orthe full length cDNA can cloned into any one of a variety of expressionvectors such as those that provide a 6His tag at the carboxyl-terminus(e.g. pCDNA 3.1 myc-his, InVitrogen).

In a typical embodiment, an expression vectors construct with a 6His tagat the carboxyl-terminus is transfected into 293T cells which are thenlabeled for one hour with ³⁵S-methionine. The cells are then washed andincubated in non-radioactive media to chase the labeled proteins forvarious time points. 20P2H8-His tagged protein is thenimmunoprecipitated using anti-His antibodies (Santa Cruz) from cellextracts and from cell supernatant (media) at various time points afterthe chase. The immunoprecipitates are analyzed by SDS-PAGE withsubsequent autoradiography to visualize ³⁵S-methionine labeled protein.

Additional embodiments of typical constructs are provided below.

Protein Expression

In an illustrative embodiment describing the production of recombinant20P2H8, 20P2H8 cDNA was cloned into the mammalian expression vectorpCDNA 3.1 (Invitrogen) that contains a 6×His COOH-terminal epitope tagthat allows protein expression analysis using an anti-His pAb reagent.This construct was used to transfect 293T human embryonic kidney cellsto assess 20P2H8 protein expression. As seen in FIG. 7 (panel A), ananti-His immunoreactive band of approximately 60 kD is seen in 293Tcells transiently transfected with the 20P2H8 vector but not in cellstransfected with a control empty vector. The molecular weightcorresponds to the predicted 57 kD molecular weight of 20P2H8 cDNA plusthe additional amino acids coded by the His and Myc epitope tags.Western analysis of Colo 205 cells, a 20P2H8 mRNA positive cell line,with an anti-20P2H8 polyclonal antibody demonstrates immunoreactivebands of approximately 58 kD and 30 kD that are not seen in 20P2H8 mRNAnegative 293T cells, which is indicative of endogenous 20P2H8 proteinexpression (FIG. 7, panel B).

Additional constructs for recombinant expression are provided below.

pGEX Constructs

To express 20P2H8 in bacterial cells, a portion of 20P2H8 was fused tothe Glutathione S-transferase (GST) gene by cloning into pGEX-6P-1(Amersham Pharmacia Biotech, NJ). All constructs were made to generaterecombinant 20P2H8 protein sequences with GST fused at the N-terminusand a six histidine epitope at the C-terminus. The six histidine epitopetag was generated by adding the histidine codons to the cloning primerat the 3′ end of the ORF. A PreScission™ recognition site permitscleavage of the GST tag from 20P2H8. The ampicillin resistance gene andpBR322 origin permits selection and maintenance of the plasmid in E.coli. In a specific illustrative embodiment, the following fragment of20P2H8 was cloned into pGEX-6P-1: Amino acids 1 to 126 (SEQ ID NO: 2).

Additional constructs can be made in pGEX-6P-1 spanning any one of avariety of regions within the 20P2H8 protein including, for example aregion containing one or more of the specified biological motifsdiscussed above or the following regions of the 20P2H8 protein: aminoacids 1 to 517; amino acids 126 to 252 and; amino acids 252 to 517 (SEQID NO: 2).

pMAL Constructs

To express 20P2H8 in bacterial cells, portions of 20P2H8 can be fused tothe maltose-binding protein (MBP) gene by cloning into pMAL-c2X andpMAL-p2X (New England Biolabs, Mass.). All constructs can be made togenerate recombinant 20P2H8 protein sequences with MBP fused at theN-terminus and a six histidine epitope at the C-terminus. The sixhistidine epitope tag was generated by adding the histidine codons tothe 3′ cloning primer. A Factor Xa recognition site permits cleavage ofthe GST tag from 20P2H8. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds. These constructs can be made in pMAL-c2X and pMAL-p2Xand span the following regions of the 20P2H8 protein: amino acids 1 to126; amino acids 1 to 517; amino acids 126 to 252; amino acids 252 to517 (SEQ ID NO: 2).

pcDNA3.1/MycHis Construct

To express 20P2H8 in mammalian cells, the 1551 bp (517 amino acid)20P2H8 ORF was cloned into pcDNA3.1/MycHis_Version A (Invitrogen,Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus(CMV) promoter. The recombinant protein has the myc and six histidinesfused to the C-terminus. The pcDNA3.1/MycHis vector also contains thebovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells expressing the protein and the ampicillinresistance gene and ColE1 origin permits selection and maintenance ofthe plasmid in E. coli.

pSRa Constructs

To generate mammalian cell lines expressing 20P2H8 constitutively, the1551 bp (517 amino acid) ORF was cloned into pSRa constructs.Amphotropic and ecotropic retroviruses are generated by transfection ofpSRa constructs into the 293T-10A1 packaging line or co-transfection ofpSRa and a helper plasmid (Φ□) in 293 cells, respectively. Theretrovirus can be used to infect a variety of mammalian cell lines,resulting in the integration of the cloned gene, 20P2H8, into the hostcell-lines. Protein expression is driven from a long terminal repeat(LTR). The Neomycin resistance gene allows for selection of mammaliancells expressing the protein and the ampicillin resistance gene andColE1 origin permits selection and maintenance of the plasmid in E.coli. An additional pSRa construct was made that fused the FLAG tag tothe C-terminus to allow detection using anti-FLAG antibodies. The FLAGsequence 5′ gat tac aag gat gac gac gat aag 3′ were added to cloningprimer at the 3′ end of the ORF.

Additional pSRa constructs can be made to produce both N-terminal andC-terminal GFP and myc/6 HIS fusion proteins of the full length 20P2H8protein.

Example 5

Production of Recombinant 20P2H8 in a Baculovirus System

To generate recombinant 20P2H8 protein in a baculovirus expressionsystem, 20P2H8 cDNA is cloned into the baculovirus transfer vectorpBlueBac 4.5 (Invitrogen) which provides a His-tag at the N-terminus.Specifically, pBlueBac-20P2H8 is co-transfected with helper plasmidpBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cellsto generate recombinant baculoviris (see Invitrogen instruction manualfor details). Baculovirus is then collected from cell supernatant andpurified by plaque assay.

Recombinant 20P2H8 protein is then generated by infection of HighFiveinsect cells (InVitrogen) with the purified baculovirus. Recombinant20P2H8 protein may be detected using 20P2H8-specific antibody. 20P2H8protein may be purified and used in various cell based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for20P2H8.

Example 6

Generation of 20P2H8 Antibodies

Generation of Monoclonal antibodies (mAbs)

In a typical method of generating 20P2H8 monoclonal antibodies, aglutathione-S-transferase (GST) fusion protein encompassing the 20P2H8protein is synthesized and used as immunogen. Balb C mice are initiallyimmunized intraperitoneally with 200 μg of the GST-20P2H8 fusion proteinmixed in complete Freund's adjuvant. Mice are subsequently immunizedevery 2 weeks with 75 μg of GST-20P2H8 protein mixed in Freund'sincomplete adjuvant for a total of 3 immunizations. Reactivity of serumfrom immunized mice to full length 20P2H8 protein is monitored by ELISAusing a partially purified preparation of HIS-tagged 20P2H8 proteinexpressed from 293T cells. Mice showing the strongest reactivity arerested for 3 weeks and given a final injection of fusion protein in PBSand then sacrificed 4 days later. The spleens of the sacrificed mice arethen harvested and fused to SPO/2 myeloma cells using standardprocedures (Harlow and Lane, 1988). Supernatants from growth wellsfollowing HAT selection are screened by ELISA and Western blot toidentify 20P2H8 specific antibody producing clones.

The binding affinity of a 20P2H8 monoclonal antibody may be determinedusing standard technology. Affinity measurements quantify the strengthof antibody to epitope binding and may be used to help define which20P2H8 monoclonal antibodies are preferred for diagnostic or therapeuticuse. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants.

Generation of Polyclonal Antibodies (pAbs)

To generate polyclonal sera to 20P2H8, a peptide was synthesizedcorresponding to amino acids 224-237 (QNALRKHKDLLGKR) of 20P2H8 proteinsequence. The peptide was coupled to Keyhole limpet hemacyanin (KLH) andused to immunize a rabbit as follows. The rabbit was initially immunizedwith 200 μg of peptide-KLH mixed in complete Freund's adjuvant Therabbit was then injected every two weeks with 200 μg of peptide-KLH inincomplete Freund's adjuvant. Bleeds were taken approximately 7-10 daysfollowing each immunization. ELISA and Western blotting analyses wereused to determine specificity and titer of the rabbit serum to theimmunizing peptide and the 20P2H8 protein respectively. Affinitypurified 20P2H8 polyclonal antibodies were prepared by passage of crudeserum from immunized rabbit over an affinity matrix comprised of 20P2H8peptide covalently coupled to Affigel 10 (BioRad). After extensivewashing of the matrix with PBS, antibodies specific to 20P2H8 peptidewere eluted with low pH glycine buffer (0.1 M, pH 2.5). Western blottingreveals the appearance of novel anti-20P2H8 immunoreactive bands ofapproximately 58 kD and 30 kD in 20P2H8 mRNA-expressing COLO 205 cellsbut not in 20P2H8 mRNA-negative 293T cells (FIG. 7, panel B).

Example 7

Identification of Potential Signal Transduction Pathways

To determine whether 20P2H8 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressing20P2H8. These transcriptional reporters contain consensus binding sitesfor known transcription factors which lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof there associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK;    growth/differentiation/apoptosis-   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

20P2H8-mediated effects may be assayed in cells showing mRNA expression,such as the 20P2H8-expressing cancer cell lines shown in FIG. 4.Luciferase reporter plasmids may be introduced by lipid mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellsextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

The transcriptional activity of 20P2H8 may be confirmed usingelectromobility shift assays (EMSA). Cells expressing 20P2H8 will beevaluated for their ability to bind known response elements and comparedto control 20P2H8 negative cells. Whole cell and nuclear extracts willbe used in this assay, and will be analyzed in the presence and absenceof potential (i) 20P2H8 inhibitors and (ii) 20P2H8 interacting proteins.These assays will provide us with valuable information regarding genecandidates and biologic pathways regulated by 20P2H8 and the mechanismby which 20P2H8 controls gene expression.

Example 8

In Vitro Assays for Characterizing 20P2H8

Subcellular Localization of 20P2H8. Sequence analysis of 20P2H8 revealedthe presence of RNP like domains and suggests that 20P2H8 may have anRNA splicing function. This, in turn, indicates that 20P2H8 may have anuclear localization. The cellular location of 20P2H8 can be assessedusing subcellular fractionation techniques widely used in cellularbiology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). Prostate,colon or pancreatic cells can be lysed and separated into nuclear,cytosolic and membrane fractions. The expression of 20P2H8 in thedifferent fractions can be tested using western blotting techniques.

Example 9

In Vitro Assays Of 20P2H₈Function. 20P2H8 function can be assessed inmammalian cells using in vitro approaches. For mammalian expression,20P2H8 can be cloned into a number of appropriate vectors, includingpcDNA 3.1 myc-His-tag and the retroviral vector pSRαtkneo (Muller etal., 1991, MCB 11:1785). Using such expression vectors, 20P2H8 can beexpressed in several cancer cell lines, including for example PC-3, NIH3T3, LNCaP and 293T. Expression of 20P2H8 can be monitored usinganti-20P2H8 antibodies.

Mammalian cell lines expressing 20P2H8 can be tested in several in vitroand in vivo assays, including cell proliferation in tissue culture,activation of apoptotic signals, primary and metastatic tumor formationin SCID mice, and in vitro invasion using a membrane invasion culturesystem (MICS) (Welch et al., Int. J. Cancer 43: 449-457). 20P2H8 cellphenotype is compared to the phenotype of cells that lack expression of20P2H8.

Cell lines expressing 20P2H8 can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and 20P2H8 overexpressing PC3, 3T3 and LNCaPcells. To assay whether 20P2H8 has chemoattractant properties, parentalindicator cells are monitored for passage through the porous membranetoward a gradient of 20P2H8 conditioned media compared to control media.This assay may also be used to qualify and quantify specificneutralization of the 20P2H8 induced effect by candidate cancertherapeutic compositions.

The function of 20P2H8 can be evaluated using anti-sense RNA technologycoupled to the various functional assays described within this section,e.g. growth, invasion and migration. Anti-sense RNA oligonucleotides canbe introduced into 20P2H8 expressing cells, thereby preventing theexpression of 20P2H8. Control and anti-sense containing cells can beanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effect of theloss of 20P2H8 expression can be evaluated. In addition to confirmingthe function of 20P2H8, anti-sense oligonucleotides may be used as atherapeutic agent.

Example 10

In Vivo Assay for 20P2H8 Tumor Growth Promotion

The effect of the 20P2H8 protein on tumor cell growth may be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×10⁶ of a number ofprostate, colon or pancreatic cell lines containing tkNeo empty vectoror 20P2H8. At least two strategies may be used: (1) Constitutive 20P2H8expression under regulation of an LTR promoter, and (2) Regulatedexpression under control of an inducible vector system, such asecdysone, tet, etc. Tumor volume is then monitored at the appearance ofpalpable tumors and followed over time to determine if 20P2H8 expressingcells grow at a faster rate. Additionally, mice may be implanted with1×10⁵ of the same cells orthotopically to determine if 20P2H8 has aneffect on local growth in the target tissue (i.e., prostate) or on theability of the cells to metastasize, specifically to lungs, lymph nodes,liver, bone marrow, etc. In relation to prostate cancer, the effect of20P2H8 on bone tumor formation and growth may be assessed by injectingprostate tumor cells intratibially, as described in WO98/16628.

These assays are also useful to determine the 20P2H8 inhibitory effectof candidate therapeutic compositions, such as for example, 20P2H8antibodies, 20P2H8 antisense molecules and ribozymes.

Example 11

In Vitro Assay of 20P2H8 Protein Interaction

Cell lines expressing 20P2H8 can also be used to identifyprotein-protein interactions mediated by 20P2H8. The presence ofproline-rich regions and homology to RNP in the ORF suggest that 20P2H8may interact with other RNPs. This possibility can be examined usingimmunoprecipitation techniques as shown by others (Hamilton B J, et al.Biochem. Biophys. Res. Commun. 1999, 261:646-51). 20P2H8 protein can beimmunoprecipitated from 20P2H8 expressing prostate cancer cell lines andexamined for protein association by western blotting. Proteininteraction may be also studied by a two yeast hybrid system, asdescribed by Shnyreva et. Al. (Shnyreva M et al, J. Biol. Chem. 2000.19;275;15498-503). These assays may also be used to analyze the effectof potential cancer therapeutics on 20P2H8 function.

To determine the contribution of the various domains contained withinthe 20P2H8 ORF to 20P2H8 function, 20P2H8 mutants can be generatedlacking one or more domains. Cell lines expressing mutant 20P2H8 proteinwill be evaluated for alteration in proliferation, invasion, migration,transcriptional activation and protein-protein interaction.

Example 12

Chromosomal Localization of 20P2H8

The chromosomal localization of 20P2H8 was determined using theGeneBridge4 radiation hybrid panel (Walter et al., 1994, Nat. Genetics7:22) (Research Genetics, Huntsville Ala.). The following PCR primerswere used to localize 20P2H8:

-   -   20P2H8.5 tcttgaaacctccagacacaagaa (SEQ ID NO: 15)    -   20P2H8.6 aagttacgatttggcttcactgg (SEQ ID NO: 16)

The resulting mapping vector for the 93 radiation hybrid panel DNAs was:

-   0000001001001010000200001101000000010100101011100010000101011010000210111000    00001101010000001

This the mapping program which can be found at internet addresshttp://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl maps 85P1B3 tochromosome 8q22.2-23.1/15q22.32-23. As HTGS hits show PACs on chromosome15 therefore this is the most likely mapping location.

Throughout this application, various publications are referenced. Thedisclosures of these publications ate hereby incorporated by referenceherein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLE 1 Start Score (Estimate of half time Rank Position SubsequenceResidue Listing of disassociation) 1 304 FLGEFATDI (SEQ ID NO: 17) 747.72 215 VLFACEEYA (SEQ ID NO: 18) 84.2 3 233 LLGKRYIEL (SEQ ID NO: 19)54.5 4 275 QQFVPPTNV (SEQ ID NO: 20) 26.1 5 363 CSAEEMNFV (SEQ ID NO:21) 23.6 6 159 FLSKENQVI (SEQ ID NO: 22) 19.7 7 450 SLGYFPTAA (SEQ IDNO: 23) 18.9 8 509 TLPKEWVCI (SEQ ID NO: 24) 17.7 9 393 YTFPAPAAV (SEQID NO: 25) 16.4 10 492 YQYATEDGL (SEQ ID NO: 26) 15.6

1. A method of diagnosing the presence of cancer in an individualcomprising: (a) determining the level of 20P2148 (SEQ ID NO: 1) mRNAexpression in a test sample obtained from the individual; and (b)comparing the level so determined to the level of 20P2118 (SEQ ID NO: 1)mRNA expression in a known normal tissue sample of the same tissue typeas the test sample, wherein elevated 20132118 (SEQ ID NO: 1) mRNAexpression in the test sample relative to the normal tissue sample isdiagnostic of the presence of cancer, and wherein the 20P2118 mRNAexpression is determined utilizing SEQ ID NO:1 as a probe.